Safety Assessment of Rosmarinus Officinalis (Rosemary)-Derived

Safety Assessment of

Rosmarinus Officinalis (Rosemary)-Derived Ingredients

as Used in Cosmetics

Status: Final Report

Release Date: June 23, 2014

Panel Meeting Date: June 9-10, 2014

The 2014 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.; Donald V.

Belsito, M.D.; Ronald A. Hill, Ph.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; James G. Marks, Jr., M.D.; Ronald

C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR Director is Lillian J. Gill, D.P.A.

This safety assessment was prepared by Monice M. Fiume, Assistant Director/Senior Scientific Analyst.

1620 L Street, NW, Suite 1200♢ Washington, DC 20036 ♢ ph 202.331.0651 ♢ fax 202.331.0088 ♢

cirinfo@cir-safety.org

ABSTRACT

The Expert Panel assessed the safety of 10 Rosmarinus officinalis (rosemary)-derived ingredients and

concluded these ingredients are safe as used in cosmetics when formulated to be non-sensitizing. The

Rosmarinus officinalis-derived ingredients are most frequently reported to function in cosmetics as skin

conditioning agents or as fragrance ingredients. The Panel reviewed the available animal and clinical

data to determine the safety of these ingredients. Because final product formulations may contain multiple

botanicals, each containing similar constituents of concern, formulators are advised to be aware of these

constituents and to avoid reaching levels that may be hazardous to consumers. Industry should use good

manufacturing practices to limit impurities that could be present in botanical ingredients.

INTRODUCTION

This report reviews the use and safety data of the following 10 Rosmarinus officinalis (rosemary)-derived ingredients as used

in cosmetics:

Rosmarinus Officinalis (Rosemary) Extract

Rosmarinus Officinalis (Rosemary) Flower Extract

Rosmarinus Officinalis (Rosemary) Flower/Leaf Stem Extract

Rosmarinus Officinalis (Rosemary) Flower/Leaf/Stem Water

Rosmarinus Officinalis (Rosemary) Leaf

Rosmarinus Officinalis (Rosemary) Leaf Extract

Rosmarinus Officinalis (Rosemary) Leaf Oil

Rosmarinus Officinalis (Rosemary) Leaf Powder

Rosmarinus Officinalis (Rosemary) Leaf Water

Rosmarinus Officinalis (Rosemary) Water

Most of the ingredients included in this review are extracts, oils, powders, or waters derived from a defined part of the

Rosmarinus officinalis (rosemary) plant.

Rosmarinus officinalis (rosemary)-derived ingredients are reported to have a number of functions, and the most common

functions in cosmetics are as a skin conditioning agent or use as a fragrance ingredient.

Two of the ingredients, i.e., rosmari-

nus officinalis (rosemary) flower extract and rosmarinus officinalis (rosemary) leaf extract, are reported to function as antiox-

idants. However, rosmarinus officinalis (rosemary) leaf powder is reported to function only as a flavoring agent.

CHEMISTRY

Definition

The definition of each Rosmarinus officinalis (rosemary)-derived ingredient indicates what part(s) of the plant from which

the ingredient is obtained (Table 1). In some cases, the definition also gives insight as to the method of manufacture.

General Characterization

The Rosmarinus officinalis L. plant, from the botanical family Lamiaceae, is a scented, evergreen shrub with a very pungent

odor that is native to the Mediterranean region and Portugal; the odor is sometimes defined as camphor-like.

2,3

Rosemary has

a spicy, harsh, bitter, aromatic taste. Bluish labiate flowers grow on the upper green part of the branches. Rosemary oil is

produced mostly in Spain, France, and Tunisia.

Rosmarinus officinalis L. is generally recognized as safe (GRAS) in foods as a spice and as a natural seasoning and flavoring.

(21CFR182.10) Rosemary has traditional or folk medicine uses, some with reported side effects.

2,5,6

The flowering dried

twig tips, the dried leaves, the fresh leaves, the fresh aerial parts, and the flowering branches are considered to be the

medicinal parts.

Chemical and Physical Properties

Rosmarinus officinalis (rosemary)-derived ingredients are strongly aromatic. Chemical and physical property data are

provided in Table 2.

Preparation/Extraction

Food-grade rosmarinus officinalis (rosemary) extract is prepared by extraction from the leaves of Rosmarinus officinalis.

Food-grade acetone, ethanol, hexane, or a combination of hexane and ethanol (in a two-step process) are used as extraction

solvents; the ethanol extract is sometimes deodorized or partially deodorized ethanol.

7,8

Food-grade rosmarinus officinalis

(rosemary) extract may also be extracted using supercritical carbon dioxide (CO

). Subsequent production steps include

filtration, purification, solvent evaporation, drying, and sieving; the extract may be deodorized, decolorized, and standardized

using diluents and carriers that are permitted in foods.

An additional method of manufacturing the cosmetic ingredients includes extraction with absolute ethanol (resulting in what

has been called “an absolute”) or a collection of the insoluble waxes (resulting in what has been called “a concrete”).

Both rosmarinus officinalis (rosemary) leaf extracts and rosmarinus officinalis (rosemary) leaf oil can be produced by

supercritical fluid extraction with natural CO

and a small amount of ethanol as a solvent.

10-13

One supplier of the leaf

extract reported that the essential oil is removed by multistep separation,

and a supplier of the leaf oil adds a small amount

(<4%) of sunflower oil to increase solubility when blending.

Food-grade rosmarinus officinalis (rosemary) leaf oil is the volatile oil obtained by steam distillation from the fresh flowering

tops or dried crushed aerial parts of Rosmarinus officinalis L.

The oil from Rosmarinus officinalis can also be obtained by

hydrodistillation of dried crushed aerial parts.

Essential oils prepared by a steam distillation process yields two distinct

fractions, a water-insoluble fraction and a water-soluble fraction.

The water-insoluble fraction contains the term oil in the

name and the water-soluble fraction contains the term water in the name. Therefore, rosmarinus officinalis (rosemary) leaf

water is the water-soluble fraction of the steam distillation of Rosmarinus officinalis (rosemary) leaves.

Constituents/Impurities

Rosmarinus officinalis L. is composed of an array of constituents, primarily phenolic acids, flavonoids, monoterpenes, diter-

penes, diterpenoids, and triterpenes. Structures for some of the principal components according to chemical family are

depicted in Figures 1-5.

A detailed list of chemical constituents by plant part is presented in Table 3, and a more focused listing of constituents of

Rosmarinus officinalis is provided in Table 4. Table 5 provides composition data on three rosmarinus officinalis (rosemary)

leaf extracts, based on certificates of analysis provided by suppliers of rosmarinus officinalis (rosemary) leaf extract; these

certificates report a phenolic diterpenes content of 14 or 25%.

15-18

According to the European Cosmetic Regulations, certain fragrance allergen compounds are subject to declaration on the

label if the concentration of a specified allergen exceeds 0.001% in leave-on and 0.01% in rinse-off products.

One supplier

declared the following concentrations of allergen compounds in a rosmarinus officinalis (rosemary) leaf extract: <0.1%

linalool and <0.2% d-limonene.

The principal antioxidative components of rosmarinus officinalis (rosemary) leaf extract are the phenolic diterpenes carnosol

and carnosic acid.

The amount of carnosol and carnosic acid present in the extract varies with the method of extraction, with

levels as low as 5-7% carnosol plus carnosic acid found in rosemary extract prepared from a partially deodorized ethanol

extract of rosemary to as high as 30% carnosol plus carnosic acid in an extract prepared with supercritical carbon dioxide.

2,7

Carnosol and carnosic acid are not the only constituents that vary with extraction method. Table 6 provides a sample of the

differences in constituent profiles in rosemary leaves based on extraction method. Some of the studies summarized in this

safety assessment provided information on the amount of constituents present in the test article; when this information was

available, it is included.

The actual amount of constituents present also varies according to the stage of development, variety of plant, season harvest-

ed, and origin of the leaves.

2,8,21,22

High-performance liquid chromatography analysis of dimethyl sulfoxide (DMSO) extracts

of rosemary leaves indicated the highest accumulation rate of the phenolic diterpenes carnosic acid, carnosol, and 12-O-

methylcarnosic acid, of rosmarinic acid, and of the flavones genkwanin and isocutellarein 7-O-glucoside was found in the

young stages of plant development.

The diterpenes and rosmarinic acid, but not the flavones, were found in the flower,

stem, and root extracts at lower concentrations than in the leaves during the early stages of plant growth, but the concentra-

tion of each, except for 12-O-methylcarnosic acid, tended to increase during flowering. Rosmarinic acid concentrations in

the leaves also decreased once flowering started, while the level in the flowers was slightly increased during flowering. The

flavones acted similarly to carnosic acid.

Water and light conditions also affect the amount of the constituents found in rosemary plants; for example, highly oxidized

diterpenes increase in rosemary plants exposed to drought and high light stress.

Although it is generally accepted that the

geographical region and stage of growth affects plant composition, some researchers reported that, within one country, the

chemical composition of rosemary essential oil (plant parts not specified) did not vary with geographical region or harvest

time.

Food-grade rosmarinus officinalis (rosemary) leaf extract has acceptance criteria of not more than 3 mg/kg arsenic and 2

mg/kg lead, and not more than 8.0% loss on drying.

Food-grade rosemary leaf oil is to have not less than 8.0% borneol and

not less than 1.5% esters, calculated as bornyl acetate.

Table 7 provides toxicity and other information on some constituents of Rosmarinus officinalis (rosemary)-derived ingredi-

ents. Because formulations may contain more than one botanical ingredient, caution is urged to avoid reaching levels of

toxicity for constituents of concern in the final formulation. Industry should use good manufacturing practices to limit

impurities.

USE

Cosmetic

The Rosmarinus officinalis (rosemary)-derived ingredients included in this safety assessment have a variety of functions in

cosmetics (Table 1). Most of the ingredients function as a skin conditioning agent and/or as a fragrance ingredient; rosmari-

nus officinalis (rosemary) leaf powder is reported to function only as a flavoring agent.

The Food and Drug Administration (FDA) collects information from manufacturers on the use of individual ingredients in

cosmetics as a function of cosmetic product category in its Voluntary Cosmetic Registration Program (VCRP). VCRP data

obtained from the FDA in 2014,

and data received in response to a survey of the maximum reported use concentration by

category conducted by the Personal Care Products Council (Council)

27,28

in 2013, indicate that nine of the ten ingredients in-

cluded in this safety assessment are currently used in cosmetic formulations (Table 8). Rosmarinus officinalis (rosemary) leaf

extract has the greatest number of uses, 729, followed by rosmarinus officinalis (rosemary) leaf oil, 474 uses, and rosmarinus

officinalis (rosemary) extract, 404 uses. According to the results of the concentration of use survey, most cosmetic formula-

tions contain very low concentrations of the Rosmarinus officinalis (rosemary)-derived ingredients, often much less than

0.1%. However, rosmarinus officinalis (rosemary) leaf extract is reported to be used at up to 10% in body and hand products

and 3% in eye shadow formulations and bath soaps and detergents. Rosmarinus officinalis (rosemary) flower/leaf/stem water

is the only ingredient not reported to be used.

In some cases, reports of uses were received in the VCRP, but concentration of use data were not provided. For example,

rosmarinus officinalis (rosemary) flower extract is reported to be used in 32 cosmetic formulations, but no use concentration

data were reported. In other cases, no uses were reported in the VCRP, but concentration of use data were received from

industry; rosmarinus officinalis (rosemary) flower/leaf/stem extract had no reported uses in the VCRP, but a use concentra-

tion in a deodorant was provided in the industry survey. Therefore, it should be presumed there is at least one use in a

deodorant formulation.

Products containing rosmarinus officinalis (rosemary)-derived ingredients may be applied to baby skin (e.g., 0.012% rosmari-

nus officinalis (rosemary) leaf extract in baby lotion, oils and creams), used in products that could be incidentally ingested

(e.g., 0.012% rosmarinus officinalis (rosemary) leaf in lipstick formulations), or used near the eye area (e.g., up to 3% ros-

marinus officinalis (rosemary) leaf extract in eye shadow formulations) or mucous membranes (e.g., up to 3% rosmarinus

officinalis (rosemary) leaf extract in bath soaps and detergents).

Additionally, Rosmarinus officinalis (rosemary)-derived

ingredients are used in cosmetic sprays and powders; for example, rosmarinus officinalis (rosemary) leaf extract is reported

to be used in other fragrance preparations at up to 0.5% and rosmarinus officinalis (rosemary) extract is used in face powders

at up to 0.05%. These products could possibly be inhaled. In practice, 95 to 99% of the droplets/particles released from

cosmetic sprays have aerodynamic equivalent diameters >10 µm.

29-32

Therefore, most droplets/particles incidentally inhaled

from cosmetic sprays would be deposited in the nasopharyngeal and bronchial regions and would not be respirable (i.e., they

would not enter the lungs) to any appreciable amount.

29,32

Rosmarinus officinalis (rosemary) extract is used in aerosol deodorants at concentrations up to 0.012%. There is some

evidence indicating that deodorant spray products can release substantially larger fractions of particulates having aerodynam-

ic equivalent diameters in the range considered to be respirable.

However, the information is not sufficient to determine

whether significantly greater lung exposures result from the use of deodorant sprays, compared to other cosmetic sprays.

All of the ingredients named in this safety assessment are listed in the European Union inventory of cosmetic ingredients.

Non-Cosmetic

Rosmarinus officinalis L. is GRAS as a spice and as a natural seasoning and flavoring when the intended use is for human

consumption (21CFR182.10) and for animal drugs, feed, and related products (21CFR582.10). It is also GRAS as an

essential oil, oleoresin (solvent-free), and natural extractive (including distillates) for human consumption (21CFR182.20)

and for animal drugs, feed, and related products (21CFR582.20). Rosemary oil can be used in the formulation of denatured

alcohol and rum (27CFR21.65).

According to The Official Journal of the European Union, extracts of rosemary contain several anti-oxidant compounds, and

although the European Food Safety Authority (EFSA) was not able to establish an acceptable daily intake due to insufficient

toxicological data, the EFSA considered the margin of safety was high enough to conclude that dietary exposure was not a

concern.

Extracts of rosemary are allowed in various food products at amounts of 30-1000 mg/kg, expressed as the sum of

carnosol and carnosic acid.

Rosemary leaves are used as a seasoning in cooking.

Rosmarinus officinalis (rosemary) leaf oil is used as a condiment and

flavoring agent in food; as an antioxidant in edible oils, meats, and other fat-containing foods; and as a dietary supplement.

Also, rosemary oil is reported to have antimicrobial activities.

Anti-inflammatory, antioxidant, and anti-microbial uses have been reported for rosemary.

21,36-38

Rosemary has traditional or

folk medicine uses, some with negative reported side effects.

2,5,6

Rosemary has been used as an antispasmodic in renal colic

and dysmenorrhea, and it has been used for relieving respiratory disorders. The essential oil is used internally as a carmina-

tive and as an appetite stimulant; however, large amount of the oil are reported to cause gastroenteritis and nephritis. The

essential oil is added to bath water as a circulation stimulant. As the oil or as an ointment, external application use is as an

analgesic liniment for rheumatism. Rosemary is used as a poultice for poorly healing wounds and in the treatment of eczema.

Additional folk medicine practices include use in lotions to treat baldness,

and use of the leaves and branches in treating

headaches.

TOXICOKINETICS

Penetration Enhancement

The effect of rosemary oil on the permeation of aminophylline was determined in human skin in vivo using attenuated total

reflection Fourier transform infrared (ATR-FTIR) spectroscopy.

Rosemary oil did enhance the permeation of aminophyl-

line; however, the increase in permeation was less than that observed with 50% ethanol.

TOXICOLOGICAL STUDIES

Single Dose (Acute) Toxicity

The acute toxicity of Rosmarinus officinalis (rosemary)-derived ingredients is not very remarkable (Table 9).

8,22,40-42

. The

dermal LD

of rosmarinus officinalis (rosemary) leaf oil is > 10 ml/kg.

The oral LD

of rosmarinus officinalis (rosemary)

leaves is >2 g/kg,

of rosmarinus officinalis (rosemary) leaf extract is >8.5 g/kg,

and of rosmarinus officinalis (rosemary)

leaf oil is 5.5 g/kg bw.

Repeated Dose Toxicity

A number of oral repeated-dose toxicity studies were performed in mice and in rats with rosmarinus officinalis (rosemary)

leaves extracted in a number of solvents (Table 10). Doses as high as 14.1 g/kg bw rosmarinus officinalis (rosemary) leaf

extract were tested (5 days by gavage), and some studies were performed for up to 3 mos (dietary) with doses of up to 400

mg/kg bw/day.

Increases in absolute and relative liver-to-body weights were observed in many of the studies, independent

of the extraction method; these changes were shown to be reversible, and no other signs of toxicity were observed. Oral

administration of rosmarinus officinalis (rosemary) leaf oil with carbon tetrachloride, but not without, resulted in an increase

in liver weights.

Ocular Irritation

Rosemary oil is reported to be a moderate ocular irritant.

(Details not provided.)

Anti-Inflammatory Effects

Rosmarinus Officinalis (Rosemary) Leaf Extract

Rosmarinus officinalis (rosemary) leaf extract has been shown to inhibit formaldehyde-induced plantar edema and 12-tetra-

decanoylphorbol 13-acetate (TPA)-induced and arachidonic acid-induced ear edema.

43,44

In the formaldehyde-induced plantar edema study, groups of six male Balb/C mice were given an injection of 20 µl of 3%

formaldehyde into the sub-plantar region of both hind paws.

After 2 h, one hind paw was treated with 10 µl of 12 mg/ml of

an ethanol extract of Rosmarinus officinalis (rosemary) leaves topically, as an injection, or both. The mice were killed after

24 h. Topical administration of the extract reduced edema by 80%, injection reduced it by 22%, and the combined applica-

tion reduced edema by 24%.

The TPA-induced ear edema study was conducted in groups of 10 male Balb/c mice.

The effect of pretreatment with 10-

1000 µg/cm

of an ethanol extract of Rosmarinus officinalis (rosemary) leaves at 30 min prior to induction of inflammation

with 25ng/cm

TPA was evaluated. The mice were killed after 4 h. Doses of 100, 250, 500, and 1000 µg/cm

of the extract

statistically significantly reduced inflammation by 38, 79, 84, and 99%, respectively.

In a TPA-induced mouse ear edema study conducted in groups of six to 10 female CD-1 mice, a single dose of 20 µl acetone,

0.5 nmol TPA, or TPA and 0.04, 0.12, or 0.36 mg of a methanol extract of Rosmarinus officinalis (rosemary) leaves in 20 µl

acetone was applied to one ear of each mouse.

The mice were killed after 5 h, and rosmarinus officinalis (rosemary) leaf

extract inhibited TPA-induced inflammation by 17, 75, and 92% respectively. The extract also inhibited TPA-induced

erythema.

In the arachidonic acid-induced mouse ear edema study, 0.02, 0.09, and 0.45 mg of a methanol extract of Rosmarinus offici-

nalis (rosemary) leaves in 20 µl acetone was applied to groups of 10 female CD-1 mice at 30 min prior to treatment with 0.3

mg arachidonic acid in 20 µl acetone.

The mice were killed after 1 h. Inflammation was inhibited by 12, 28, and 54%,

respectively.

Effect on Epidermal Hyperplasia

Two-hundred µl acetone, 1 nmol TPA, or 1 nmol TPA and 3.6 mg rosmarinus officinalis (rosemary) leaf extract in 200 µl

acetone were applied twice a day for 4 days to the dorsal skin of mice.

Three or four CD-1 mice were used per group.

Topical application of the extract with TPA inhibited a TPA-induced increase in the number of epidermal cell layers and

epidermal thickness.

Immunologic Effects

An aqueous extract of up to 2.5 mg/ml Rosmarinus officinalis (rosemary) leaves was found to inhibit ultraviolet (UV)-

induced up-regulation of matrix metalloproteinase-1 (MMP-1) gene transcription in dermal human fibroblasts.

The release

of the cytokines interleukin (IL)-1α and IL-6 was prevented by the extract.

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

Non-Human

Rosmarinus Officinalis (Rosemary) Leaf Extract

Oral administration of high doses of rosmarinus officinalis (rosemary) leaf extract adversely affected fertility in male rats.

Groups of 10 male Sprague Dawley rats were fed a diet with 0, 250 or 500 mg/kg bw/day of an ethanol extract of Rosmarinus

officinalis (rosemary) leaves in distilled water. After 53 days of dosing, each male rat was mated with two untreated female

rats for 10 days; the female rats had been given a subcutaneous (s.c.) dose of 5.0 mg estradiol benzoate 54 h and 0.5 mg

progesterone at 54 and 6 h, respectively, prior to being placed with the males. The males were dosed during, and killed after,

the 10-day mating period, and the reproductive organs were examined. The females were killed 1 wk after the mating period,

and the reproductive tract of each female was examined to determine pregnancy and the number of implantation sites, viable

fetuses, and fetal resorptions.

Body weights of the male rats of the test groups were similar to those of the control group. However, the high dose group

exhibited statistically-significantly reduced absolute weights and organ-to-body weight ratios of testes and male accessory

sex organs, diameters of seminiferous tubules and Leydig cell nuclei, height of epithelia of the epididymes and seminal

vesicles, germinal and interstitial cell counts, levels of sex hormones, and sperm density and motility when compared to the

controls. The numbers of interstitial degenerating cells were statistically-significantly increased in the high-dose group.

Exposure of the males to the high dose resulted in a reduced number of pregnant females, implantations and viable fetuses,

and an increased the number of resorptions. Results from the low-dose groups suggested dose-response trends in these

parameters, although statistically-significant differences were observed only with the high-dose group.

Rosmarinus Officinalis (Rosemary) Flower/Leaf/Stem Extract

A group of 12 gravid female Wistar rats was dosed by gavage with 26 mg/day of a 30% aq. extract of rosmarinus officinalis

(rosemary) flower/leaf/stem extract (13 mg/ml solids) on days 1-6 of gestation (preimplantation), and a group of 14 gravid

rats was dosed with the extract on days 6-15 of gestation (organogenesis).

Negative control groups of 12 or 11 gravid rats

were given saline by gavage on days 1-6 or 6-15 of gestation, respectively. All dams were killed on day 21 of gestation. No

signs of maternal toxicity were observed, and maternal weight gains were similar for treated and control groups.

In the rats dosed on days 1-6 of gestation, a non-statistically significant increase in preimplantation loss was observed. No

changes in post-implantation loss were seen as compared to controls, and no other reproductive parameters were affected. In

the group treated on days 6-15 of gestation, a non-statistically significant increase in post-implantation loss rate (2.54%) was

reported; analysis of the resorptions found that they occurred during the early post-implantation period. No other changes in

reproductive parameters were observed when compared to the negative control group. Developmental effects were not ob-

served in either group.

Human

According to the PDR for Herbal Medicines, rosemary preparations should not be used as a drug during pregnancy; very

large quantities of the leaves reportedly can be misused as an abortifacient.

According to Herbal Drugs and Phytopharma-

ceuticals, toxic side effects may occur with components of the essential oil.

(Details were not provided.)

Effects on Estrogenic Activity

Non-Human

Rosmarinus Officinalis (Rosemary) Leaf Extract

Groups of seven or eight 6-wk old ovariectomized CD-1 mice were fed either a diet containing 2% of a methanol extract of

Rosmarinus officinalis (rosemary) leaves or the basal diet.

After 3 wks, the animals were given an i.p. injection of 0, 45, or

100 ng/mouse estradiol or estrone in 50 µl corn oil, once daily for 3 days. Eighteen hours after the last injection, the animals

were killed and the uterus was removed. In the mice fed the basal diet, estradiol and estrone increased the uterine wet weight

in a dose-dependent manner. Rosemary inhibited 35-50% of the uterine response; this was statistically significant.

Human

Rosmarinus Officinalis (Rosemary) Leaf Extract

In a study investigating the effects of a botanical supplement on sex steroid hormones and metabolic markers in premeno-

pausal women, a few changes were found, however, the changes were not very remarkable.

A group of 15 premenopausal

women were asked to take a supplement containing 100 mg Rosmarinus officinalis (rosemary) leaf 5:1 extract; 100 mg Cur-

curma longa (turmeric) root extract standardized to 95% curcumin; 100 mg Cyanara scolymus (artichoke) leaf 6:1 extract;

100 mg Silybum marinum (milk thistle) seed extracted; 100 mg Taraxacum officinalis (dandelion) root 4:1 extract; and 50 mg

Schidandra chinensis (berry) 20:1 extract. Four capsules were to be taken twice a day with meals. Rice powder placebo

capsules were given to a group of 15 premenopausal women using the same dosing regimen. Blood and urine samples were

collected during the early-follicular and mid-luteal phases of study menstrual cycles 1 and 5.

On average, test subjects took 6.3 capsules/day, and controls took 7.1 capsules/day. Compared to the placebo group, the fol-

lowing changes from Cycle 1 to Cycle 5 in early-follicular phase serum hormone concentrations were statistically significant

or borderline significant: decreases in serum dehydroepiandrosterone (-13.2%, p= 0.02); dehydroepiandrosterone sulfate (-

14.6%, p=0.07); androstenedione (-8.6%, p=0.05); and estrone sulfate (-12.0%, p=0.08). No other statistically significant

changes or trends were observed for other serum sex steroid hormones, serum metabolic markers, or urinary estrogen metab-

olites at either phase.

GENOTOXICITY

In vitro, rosemary extract (solvent not specified)

and rosmarinus officinalis (rosemary) leaf oil

were not mutagenic in an

Ames test, and rosmarinus officinalis (rosemary) leaf extract was not genotoxic in an Ames test, a chromosomal aberration

assay in human lymphocytes, or a gene-locus mutation assay in human lymphocytes

(Table 11). In in vivo studies in mice

and rats, oils that were extracted by hydrodistillation induced statistically significant increases in chromosomal aberrations

without gaps in a chromosomal aberration assay at 2000 mg/kg bw, increases in micronucleated polychromatic erythrocytes

(MNPCEs) in several micronucleus tests at 1000 and 2000 mg/kg bw, and increases in DNA damage in a comet assay at

≥300 mg/kg bw;

14,41

however, no genotoxic effects were seen in mice in a micronucleus test at 1500 mg/kg bw/day with

leaves extracted with absolute ethanol.

A hydro-alcoholic extract of rosemary was not genotoxic in a chromosomal aberra-

tion assay or a micronucleus test in rats.

A mixture containing 19% Rosmarinus officinalis (rosemary) leaves, 71.5% St.

John’s Wort, and 9.5% spirulina induced in mice statistically significant increases in MNPCEs at 760 and 1520 mg/kg

bw/day in a micronucleus test; in frequency of aneuploidy, percent polyploidy, and total percent aberrations with 760 and

1520 mg/kg bw/day in a chromosomal aberration assay; and in frequency of banana-shaped, swollen acrosome, and triangu-

lar head sperm abnormalities and percent total spermatozoa abnormalities at 1520 mg/kg bw/day in a spermatozoa abnormal-

ity assay.

Rosmarinus officinalis (rosemary) leaf extract was shown to have anti-mutagenic potential, in vitro, in an Ames test with

Salmonella typhimurium and in Comet assays in a human hepatoma cell line..

In vivo, in micronucleus assays, rosmarinus

officinalis (rosemary) leaf extract did not decrease the number of MNPCEs induced in mice by a genotoxic agent.

CARCINOGENICITY

Effects on Tumor Promotion

Topical application of methanol and double distilled water extracts of Rosmarinus officinalis (rosemary) leaves statistically

significantly decreased skin tumors in mice; in these studies, 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene

(B(a)P )was used for initiation and TPA

or croton oil

55,56

was used for promotion (Table 12). Dietary administration of

1.0% rosmarinus officinalis (rosemary) leaf extract decreased the incidence of palpable mammary tumors in rats caused by

DMBA.

IRRITATION AND SENSITIZATION

Skin Irritation/Sensitization

An ointment containing 4.4% rosmarinus officinalis (rosemary) leaf oil (and other essential oils), applied at concentrations up

to 40%, was not irritating to rat skin (Table 13).

However, in a rabbit study, occlusive application to intact and abraded

skin produced moderate irritation.

In clinical testing, Rosmarinus officinalis (rosemary) leaves produced irritation (scores of +/-, +, or ++) in 44/234 patients

with contact dermatitis or eczema (Table 13).

A supercritical extract and the absolute of Rosmarinus officinalis (rosemary)

leaves were considered weak irritants in a small study with test populations of 20-25 subjects; the extracts were not

phototoxic.

Formulations containing up to 0.2% rosmarinus officinalis (rosemary) leaf extract were not irritants or

sensitizers.

60-62

Rosmarinus officinalis (rosemary) leaf oil, 10% in petrolatum, was not an irritant in a 48-h closed patch test,

or a sensitizer in a maximization study;

a formulation containing 1.5% rosmarinus officinalis (rosemary) leaf oil was not an

irritant or a sensitizer in an HRIPT.

Phototoxicity

Rosmarinus Officinalis (Rosemary) Leaf Extract

The phototoxicity of rosmarinus officinalis (rosemary) leaf extract extracted with supercritical CO

, as a concrete (insoluble

wax) extracted in hexane, or as a concrete extracted in hexane, was evaluated.

Photopatch tests were performed on two of

three test sites; one site was irradiated with 10 J/cm

UVA and the second site with 75% of the minimal erythema dose of

UVB. The test sites were scored after 48 and 72 h, and were compared to the non-irradiated site. None of the extracts were

phototoxic.

Case Reports

Several cases of allergic reactions to Rosmarinus officinalis (rosemary) have been reported (Table 14).

64-72

In some of the

studies, follow-up patch testing included photopatch tests; generally, reactions were stronger in the photopatch tests when

compared to standard testing.

68,69

Some of the follow-up patch testing included carnosol; testing with 0.1 and 1.0% carnosol

resulted in positive reactions.

65,69

SUMMARY

This report addresses the safety of 10 Rosmarinus officinalis (rosemary)-derived ingredients as used in cosmetics. Most of

the ingredients included in this review are extracts, essential oils, powders, or waters derived from a defined part of the

Rosmarinus officinalis (rosemary) plant. The Rosmarinus officinalis (rosemary)-derived ingredients are reported to have a

number of functions in cosmetics, and the most common functions are as a skin conditioning agent or as a fragrance ingredi-

ent. According to VCRP data obtained from the FDA, rosmarinus officinalis (rosemary) leaf extract has the most uses, 729,

followed by rosmarinus officinalis (rosemary) leaf oil, 474 uses, and rosmarinus officinalis (rosemary) extract, 404 uses.

Most of the reported use concentrations for Rosmarinus officinalis (rosemary)-derived ingredients are well below 0.1%.

However, rosmarinus officinalis (rosemary) leaf extract has higher concentrations of use reported, specifically, use at up to

10% in body and hand products and 3% in eye shadow formulations and bath soaps and detergents. Rosmarinus officinalis

(rosemary) flower/leaf/stem water is the only ingredient not reported to be used.

Rosmarinus officinalis (rosemary) extract is prepared by extraction from the leaves of Rosmarinus officinalis with acetone,

ethanol, hexane, a combination of hexane and ethanol (in a two-step process), or supercritical CO

; it can also be prepared

from a deodorized or partially deodorized ethanol extract of rosemary. Additional methods include extraction with absolute

ethanol (resulting in an absolute) or a collection of the insoluble waxes (resulting in a concrete).

Rosmarinus officinalis L. is composed of an array of constituents, primarily phenolic acids, flavonoids, monoterpenes, diter-

penes, diterpenoids, and triterpenes. The principal antioxidative components of rosmarinus officinalis (rosemary) leaf extract

are the phenolic diterpenes carnosol and carnosic acid. The actual amount of constituents present varies according to the

stage of development, variety of plant, season harvested, origin of the leaves, and extraction method.

Rosemary oil increased the permeation of aminophylline through human skin, but the increase was not as great as that seen

with 50% ethanol.

The acute toxicity of Rosmarinus officinalis (rosemary)-derived ingredients is not very remarkable. The dermal LD

of ros-

marinus officinalis (rosemary) leaf oil is > 10 ml/kg. The oral LD

of rosmarinus officinalis (rosemary) leaves is >2 g/kg,

of rosmarinus officinalis (rosemary) leaf extract is >8.5 g/kg, and of rosmarinus officinalis (rosemary) leaf oil is 5.5 g/kg bw.

A number of oral repeated-dose toxicity studies were performed in mice and in rats with Rosmarinus officinalis (rosemary)

leaves extracted in a various solvents. Doses as high as 14.1 g/kg bw rosmarinus officinalis (rosemary) leaf extract were

tested (5 days by gavage), and some studies were performed for up to 3 mos (dietary) with doses of up to 400 mg/kg bw/day.

Increases in absolute and relative liver-to-body weights were observed in many of the studies, independent of the extraction

method; these changes were shown to be reversible, and no other signs of toxicity were observed. Oral administration of

rosmarinus officinalis (rosemary) leaf oil with carbon tetrachloride, but not without, resulted in an increase in liver weights.

Rosmarinus officinalis (rosemary) leaf extract has been shown to have anti-inflammatory activity. Rosmarinus officinalis

(rosemary) leaf extract inhibited a TPA-induced increase in the number of epidermal cell layers and epidermal thickness in

mouse skin.

A high dose (500 mg/kg/day) of Rosmarinus officinalis (rosemary) leave extract was a reproductive toxicant in a dietary

study in male rats. In a study in gravid female Wistar rats, no statistically significant changes were observed after oral dosing

with 26 mg/day of a 30% aq. rosmarinus officinalis (rosemary) flower/leaf/stem extract during preimplantation or during

organogenesis. In a dietary study in ovariectomized CD-1 mice, 2% of a methanol extract of Rosmarinus officinalis (rose-

mary) leaves inhibited the uterine response in a statistically significant manner.

In a clinical study investigating the effects on sex steroid hormones and metabolic markers of a botanical supplement contain-

ing 100 mg Rosmarinus officinalis (rosemary) leaf 5:1 extract (and other botanical ingredients) in premenopausal women, a

few changes were found. Overall, the changes were not remarkable.

In vitro, rosemary extract (solvent not specified) and rosmarinus officinalis (rosemary) leaf oil were not mutagenic in an

Ames test, and rosmarinus officinalis (rosemary) leaf extract was not genotoxic in an Ames test, a chromosomal aberration

assay in human lymphocytes, or a gene-locus mutation assay in human lymphocytes. In in vivo studies in mice and rats, oils

that were extracted by hydrodistillation induced statistically significant increases in chromosomal aberrations without gaps in

a chromosomal aberration assay at 2000 mg/kg bw, increases in micronucleated polychromatic erythrocytes (MNPCEs) in

several micronucleus tests at 1000 and 2000 mg/kg bw, and increases in DNA damage in a comet assay at ≥300 mg/kg bw;

however, no genotoxic effects were seen in mice in a micronucleus test at 1500 mg/kg bw/day with leaves extracted with ab-

solute ethanol. A hydro-alcoholic extract of rosemary was not genotoxic in a chromosomal aberration assay or a micronucle-

us test in rats. A mixture containing 19% Rosmarinus officinalis (rosemary) leaves, 71.5% St. John’s Wort, and 9.5% spiru-

lina induced in mice statistically significant increases in MNPCEs at 760 and 1520 mg/kg bw/day in a micronucleus test; in

frequency of aneuploidy, percent polyploidy, and total percent aberrations with 760 and 1520 mg/kg bw/day in a chromo-

somal aberration assay; and in frequency of banana-shaped, swollen acrosome, and triangular head sperm abnormalities and

percent total spermatozoa abnormalities at 1520 mg/kg bw/day in a spermatozoa abnormality assay.

Rosmarinus officinalis (rosemary) leaf extract was shown to have anti-mutagenic potential in vitro. In vivo, in micronucleus

assays, rosmarinus officinalis (rosemary) leaf extract did not decrease the number of MNPCEs induced by a genotoxic agent.

Topical application of methanol and double distilled water extracts of rosmarinus officinalis (rosemary) leaves statistically

significantly decreased skin tumors in mice; in these studies, DMBA or benzo[a]pyrene was used for initiation and TPA or

croton oil was used for promotion. Dietary administration of 1.0% rosmarinus officinalis (rosemary) leaf extract decreased

the incidence of palpable mammary tumors in rats caused by DMBA.

An ointment containing 4.4% rosmarinus officinalis (rosemary) leaf oil (and other essential oils), applied at concentrations up

to 40%, was not irritating to rat skin. However, in a rabbit study, occlusive application to intact and abraded skin produced

moderate irritation.

In clinical testing, Rosmarinus officinalis (rosemary) leaves produced irritation (scores of +/-, +, or ++) in 44/234 patients

with contact dermatitis or eczema. A supercritical extract and the absolute of Rosmarinus officinalis (rosemary) leaves were

considered weak irritants in a small study with test populations of 20-25 subjects; the extracts were not phototoxic. Formula-

tions containing up to 0.2% rosmarinus officinalis (rosemary) leaf extract were not irritants or sensitizers. Rosmarinus

officinalis (rosemary) leaf oil, 10% in petrolatum, was not an irritant in a 48-h closed patch test, or a sensitizer in a maximi-

zation study in 25 subjects; a formulation containing 1.5% rosmarinus officinalis (rosemary) leaf oil was not an irritant or a

sensitizer in an HRIPT in 104 subjects.

Several cases of allergic reactions to Rosmarinus officinalis (rosemary) have been reported. In some of the studies, follow-up

patch testing included photopatch tests; generally, reactions were stronger in the photopatch tests, compared to standard

testing. Some also evaluated the effect of carnosol; testing with 0.1 and 1.0% carnosol resulted in positive reactions.

DISCUSSION

Upon initial review of the safety assessment of Rosmarinus officinalis (rosemary)-derived ingredients, the Panel issued an

Insufficient Data Announcement requesting the following:

1. Dermal sensitization data for 10% rosmarinus officinalis (rosemary) leaf extract (i.e., a human repeated-insult patch

test in a sufficient number of subjects at concentration of use);

2. Chemical characterization of the flower, if available;

3. Additional information on the deodorizing process performed during preparation of some of the ingredients,

including information on what by-products may form; and

4. Information as to why the PDR of Herbal Medicines states that rosemary preparations should not be used during

pregnancy.

The majority of these data were not received. Rosmarinus officinalis is GRAS as a spice, and although that alleviates the

concern of oral exposure with cosmetic use, dermal irritation and sensitization data are necessary to determine safety.

Additional information on the deodorizing process that is part of the preparation of some of the ingredients also was not

received. After further discussion, the Panel stated that because the deodorizing process is part of the preparation of food-

grade rosmarinus officinalis (rosemary) extract, and because data are included in this safety assessment on some ingredients

that were deodorized and no adverse effects were found, the Panel was not concerned with obtaining additional information

on this process or the by-products that might form.

The Panel did note that because botanical ingredients, derived from natural plant sources, are complex mixtures, there is con-

cern that multiple botanical ingredients may each contribute to the final concentration of a single constituent. Therefore,

when formulating products, manufacturers should avoid reaching levels in final formulation of plant constituents that may

cause sensitization or other adverse effects. Specific examples of constituents that could possibly induce sensitization are

linalool or monoterpenes, and those that could possibly cause adverse effects are caffeic acid and terpenes, such as thujone,

limonene, and methyleugenol.

The Panel expressed concern about pesticide residues and heavy metals that may be present in botanical ingredients. They

stressed that the cosmetics industry should continue to use current good manufacturing practices (cGMPs) to limit impurities.

One study evaluated the irritation potential of Rosmarinus officinalis (rosemary) leaves in patients with contact dermatitis or

eczema. The Panel stated that because the test subjects were patients with eczematous skin, the report of irritation could not

be interpreted for relevance to cosmetic use.

The Panel discussed the positive results observed in a reproductive toxicity study in male rats fed 500 mg/kg/day rosmarinus

officinalis (rosemary) leaf extract, as well as the caution in the PDR for Herbal Medicines stating that rosemary preparations

should not be used as a drug during pregnancy. The effects in the rat study were observed at exposure concentrations that

would be well above those used in cosmetic products, and the PDR refers to the use of rosemary as a drug at very high con-

centrations. Because these effects were observed only at very high concentrations, and because no statistically significant

effects were reported in a study in rats dosed orally with 26 mg/day of a 30% aq. extract of rosmarinus officinalis (rosemary)

flower/leaf/stem extract, reproductive and developmental toxicity is not a concern with cosmetic use of Rosmarinus

officinalis (rosemary)-derived ingredients, which are mostly used at very low concentrations.

Finally, the Panel discussed the issue of incidental inhalation exposure to Rosmarinus officinalis (rosemary)-derived ingredi-

ents. The Panel stated that although there were no inhalation data available, the Rosmarinus officinalis (rosemary)-derived

ingredients are used at very low concentrations in products that could incidentally be inhaled; e.g., rosmarinus officinalis

(rosemary) leaf extract is used in other fragrance preparations and rosmarinus officinalis (rosemary) extract is used in face

powders. The Panel noted that in aerosol products, 95% – 99% of droplets/particles would not be respirable to any appreci-

able amount. Furthermore, droplets/particles deposited in the nasopharyngeal or bronchial regions of the respiratory tract

present no toxicological concerns based on the chemical and biological properties of these ingredients. Coupled with the

small actual exposure in the breathing zone and the concentrations at which the ingredients are used, the available informa-

tion indicates that incidental inhalation would not be a significant route of exposure that might lead to local respiratory or

systemic effects. A detailed discussion and summary of the Panel’s approach to evaluating incidental inhalation exposures to

ingredients in cosmetic products is available at http://www.cir-safety.org/cir-findings

CONCLUSION

The CIR Expert Panel concluded that the following ten Rosmarinus officinalis (rosemary)-derived ingredients are safe in the

present practices of use and concentration in cosmetics described in this safety assessment when formulated to be non-

sensitizing:

Rosmarinus Officinalis (Rosemary) Extract

Rosmarinus Officinalis (Rosemary) Flower Extract

Rosmarinus Officinalis (Rosemary) Flower/Leaf/Stem Extract

Rosmarinus Officinalis (Rosemary) Flower/Leaf/Stem Water*

Rosmarinus Officinalis (Rosemary) Leaf

Rosmarinus Officinalis (Rosemary) Leaf Extract

Rosmarinus Officinalis (Rosemary) Leaf Oil

Rosmarinus Officinalis (Rosemary) Leaf Powder

Rosmarinus Officinalis (Rosemary) Leaf Water

Rosmarinus Officinalis (Rosemary) Water

*Not reported to be in current use. If this ingredient was to be used in the future, the expectation is that it would be used in product

categories and at concentrations comparable to others in this group.

FIGURES

Figure 1. Principal diterpenes

1a. Carnosol

C C

1b. Carnosic acid

C C

1c. Rosmanol

Figure 2. Principal triterpenes

C C

2a. Oleanolic acid

2b. Ursolic acid

2c. Betulin

2d. α-Amyrin

2e. β-Amyrin

Figure 3. Principal flavonoids

3a. Genkwanin

3b. Cirsimarin

3c. Luteolin

3d. Diosmetin

3e. Apigenin

Figure 4. Phenolic acids

4a. Caffeic acid

4b. Chlorogenic acid

4c. Neochlorogenic acid

4d. Labiatic acid

Figure 5. Principal Volatiles

5a. 1,8-Cineole

5b. Camphor

5c. α-Pinene

5d. Borneol

TABLES

Table 1. Definitions and reported functions

Ingredient (CAS No.)

Definition

Reported Function(s)

Rosmarinus Officinalis (Rosemary) Extract

(84604-14-8)

the extract of the whole plant Rosmarinus officinalis

skin-conditioning agent – misc

Rosmarinus Officinalis (Rosemary) Flower

Extract

the extract of the flowers of Rosmarinus officinalis

antioxidant; deodorant agents; skin-

conditioning agents – misc

Rosmarinus Officinalis (Rosemary)

Flower/Leaf/Stem Extract

the extract of the flowers, leaves and stems of

Rosmarinus officinalis

fragrance ingredients; skin-conditioning

agents - misc

Rosmarinus Officinalis (Rosemary)

Flower/Leaf/Stem Water

the aqueous solution of the steam distillates obtained

from the flowers, leaves and stems of

Rosmarinus

officinalis

fragrance ingredient

Rosmarinus Officinalis (Rosemary) Leaf

the leaf of Rosmarinus officinalis

skin-conditioning agents – misc

Rosmarinus Officinalis (Rosemary) Leaf

Extract (84604-14-8)

the extract of the leaves of Rosmarinus officinalis

antimicrobial agents; antioxidant; fragrance

ingredients; skin-conditioning agents -

miscellaneous; skin-conditioning agents –

occlusive

Rosmarinus Officinalis (Rosemary) Leaf

Oil (8000-25-7)

the essential oil obtained from the flowering tops and

leaves of Rosmarinus officinalis

fragrance ingredients; skin-conditioning

agents – misc

Rosmarinus Officinalis (Rosemary) Leaf

Powder

the powder derived from the dried, ground leaves of

Rosmarinus officinalis

flavoring agents

Rosmarinus Officinalis (Rosemary) Leaf

Water

an aqueous solution of the steam distillate obtained from

the leaves of Rosmarinus officinalis

fragrance ingredient

Rosmarinus Officinalis (Rosemary) Water

an aqueous solution of the steam distillate obtained from

Rosmarinus officinalis

fragrance ingredient

Table 2. Chemical and physical properties

Property

Description

Reference

Rosmarinus Officinalis (Rosemary) Leaf

odor

strongly aromatic

Rosmarinus Officinalis (Rosemary) Leaf Extract

physical state and appearance

powder or liquid

colorless, volatile oil

dark brown viscous liquid with a characteristic smell and taste (as the extract (and)

Helianthus Annuus Seed Oil)

10,11

solubility

insoluble in water

refractive index

1.4710 - 1.4740

density

0.9165 - 0.9220

Rosmarinus Officinalis (Rosemary) Leaf Oil

physical state and appearance

colorless or pale yellow liquid with characteristic odor and a warm, camphoraceous

taste

colorless, pale yellow, or pale green liquid with a camphorous odor

7,35

solubility

almost insoluble in water

soluble in most vegetable oils; insoluble in alcohol and in propylene glycol

density (d

)

0.894-0.912

0.907-0.920

index of refraction (n

)

1.464-1.476

Rosmarinus Officinalis (Rosemary) Leaf Powder

physical state and appearance

greyish-green to yellowish-green powder

Table 3. Chemical constituents by plant part (ppm)

Constituent*

Plant

Leaf

Flower

Shoot

Resin,

Exudate, Sap

Essential Oil

carbohydrates

640,600-

704,660

fiber

165,420-

206,338

fat

134,020-

187,418

water

77,900-108,300

ash

61,900-75,570

protein

40,700-62,568

ursolic acid

28,000-41,000

rosmarinic acid

25,000

3500

13,500

3300-25,000

calcium

10,919-16,150

potassium

8842-11,284

oleanolic acid

10,500

carnosol

530-9803

cineole

168-9728

1,8-cineole

8125

camphor

60-5800

myrcene

25-5605

bornyl acetate

5054

α –pinene

235-4750

borneol

12-4237

magnesium

2142-2483

rosmaric acid

3000-3500

camphene

23-2350

β-caryophyllene

12-2075

70-2075

toluene

436-2071

limonene

1950

α –terpineol

24-1555

β-pinene

17-1425

phosphorus

490-1000

p-cymene

25-950

carvone

16-760

α-humulene

725

salicylates

70-680

ascorbic acid

612-673

α-amorphene

70-665

γ-muurolene

70-665

phytosterols

580-640

sodium

462-592

linalool

585

α –terpinene

4-555

terpinen-4-ol

4-521

α –thujene

1-475

δ-terpineol

7-418

iron

220-400

α –thujone

84-399

(E)-β-ocimene

380

verbenone

10-375

geraniol

50-370

3-hexanone

74-351

terpinolene

12-350

caryophyllene

16-340

δ-3-carene

330

fenchone

250

β-thujone

11-209

β-elemene

3-200

sabinene

190

mesityl alcohol

40-190

linalool acetate

32-152

α –phellandrene

133

α- fenchyl alcohol

28-133

p-menth-3-en-1-ol

28-133

3,5,5-trimethylhexan-1-ol

28-133

trans-ocimene

4-130

cis-pinan-3-one

17-110

4-terpinenyl-acetate

12-110

safrole

32-95

Table 3. Chemical constituents by plant part (ppm)

Constituent*

Plant

Leaf

Flower

Shoot

Resin,

Exudate, Sap

Essential Oil

cis-β-terpineol

20-95

α- fenchyl acetate

20-95

longifolene

20-95

isoborneol

7-95

rosmanol

(+)-limonene

16-76

δ-cadinene

caryophyllene oxide

(Z)-β-ocimene

trans-pinocarveol

32-42

3-octanone

20-40

boron

22-39

zinc

30-38

AR-curcumene

8-38

methyl heptenone

8-38

myrtenol

8-38

lavandulol

7-34

trans-β-terpineol

7-34

trans-myrtenol

benzyl alcohol

7-32

elemol

7-32

γ-eudesmol

7-32

rosmadial

α-amyrenone

β-amyrenone

epirosmanol

β-carotene

19-21

rofficerone

trans-sabinene hydrate

manganese

18-19

cis-α-bisabolene

4-19

isopinocarveol

4-19

isopulegol

4-19

3-octanol

4-19

dimethyl styrene

1-19

7-methoxy-rosmanol

isorosmanol

cis-myrtenol

11-17

cisimaritrin

α-amyrin

β-amyrin

botulin

12.1

α –muurolene

2-12

3-o-acetyloleanolic acid

3-o-acetylursolic acid

niacin

10-11

peperitenone

4-8

eugenol methyl ether

5-7

copper

5-6

thiamin

5-6

carvacrol

5-6

α -terpinenyl acetate

5-6

allo-aromadendrene

4-5

neo-thujol

1.5-5

calamenene

1-5

trans-carveol

1-5

p-cymen-8-ol

1-5

nopol

1-5

γ-candinene

1-5

α-copaene

2-4

epi-

-bisabolol

sabinyl acetate

1.5

β-gurjunene

0.5

cis-sabinene hydrate

0.4

β-phellandrene

trace

tricyclene

trace

α-fenchol

trace

p-menth-cis-en-1-ol

trace

Table 3. Chemical constituents by plant part (ppm)

Constituent*

Plant

Leaf

Flower

Shoot

Resin,

Exudate, Sap

Essential Oil

p-menth-trans-en-1-ol

trace

trans-anethole

apigen-7-glucoside

betulin

bornylene

cadalene

caffeic acid

calacorene

carnosic acid

chlorogenic acid

cirsilion

cubenene

diosmetin

epi-

-amyrin

eriodictiol

ethanol

α-fenchene

β-fenchene

genkwanin-4’-methyl ether

glycolic acid

genkwanin

hesperidin

hispidulin

hispiduloside

humulene epoxide I

humulene epoxide II

5-hydroxy-4',7-dimethoxyflavone

hydroxybenzoic acid-4-

-D-glucoside

4-hydroxybenzoyl glucoside

α-hydroxyhydrocaffeic acid

-hydroxyoleanolic acid

3-β-hydroxyurea-12,20(30)-dien-17-on

acid

19-

-hydroxyursolic acid

isobornyl acetate

isobutyl acetate

isorosmaricine

labiatic acid

ledene

luteolin

luteolin-7-glucoside

6-methoxy-genkwanin

6-methoxy-luteolin

6-methoxy-luteolin-7-glucoside

6-methoxyluteolin-7-methyl ether

methyl ether

methyl eugenol

N-methyl rosmaricine

neo-chlorogenic acid

nepetin

nepetrin

1-octen-3-ol

picrosalvin

rosmadiol

rosmaricine

rosmaridiphenol

rosmarinol

rosmariquinone

salvigenin

santene

salicylic-acid-2-β-D-glucoside

α –selinene

sinensetin

β-sitosterol

squalene

syringic-acid-4-β-D-glucoside

tannin

thymol

Table 3. Chemical constituents by plant part (ppm)

Constituent*

Plant

Leaf

Flower

Shoot

Resin,

Exudate, Sap

Essential Oil

trimethylalkane

o-o-N-trimethylrosmaricine

vanillic-acid-4-β-D-glucoside

verbenol

betulinic acid

δ-4-carene

diosmin

7-ethoxy-rosmanol

luteolin-3’-o-(3”-o-acetyl)-

-D-

glucuronide

luteolin-3’-o-(4”-o-acetyl)-

-D-

glucuronide

luteolin-3’-o-

-D-glucuronide

monomethyl alkane

pristane

protocatechuic-acid-4-β-D-glucoside

pectin

acetic acid

butan-2-ol

caproic acid

deca-trans-2,trans-4-dien-1-al

hept-trans-2-en-1-al

heptan-1-al

heptan-2-ol

heptanoic acid

hexan-1-al

hexan-1-ol

3-methyl-butan-1-ol

β-ocimene

octan-1-ol

octane-2,3-dione

octanoic acid

pentan-1-al

pentan-1-ol

pentan-2-ol

zingiberene

dipentene

*constituents reported in ppm

NS – amount not specified

“ – “ means not reported

Table 4. Constituent data by plant part

Reference

Plant part not specified

- volatile oil (0.5-2.5%): 1,8-cineole (20-50%); camphor (10-25%); α-pinene (up to 25%); other monoterpenes (including borneol

and limonene)

- rosmarinic acid

- diterpene bitter substances: carnosol; carnosolic acid (picrosalvin); isorosmanol; rosmanol; rosmadiol; rosmaridiphenol

rosmariquinone

- triterpene acids: ursolic acid; oleanolic acids; rosmanol; 7-ethoxyrosmanol; betulic acid; carnosol; traces of 19α-

hydroxyursolic, 2β-hydroxyoleanolic, and 3β-hydroxyurea-12,20(30)-dien-17-oic acids

- triterpene alcohols: α-amyrin; β-amyrin; betulin

- flavonoids: luteolin; genkwanin (7-O-methlylapigenin); diosmetin; diosmin; genkwanin-4’-methyl ether; 6-methoxygenkwanin;

6-methyoxyluteolin; 6-methoxyluteolin-7-glucoside; 6-methoxyluteolin-7-methylether; hispidulin; apigenin

- corresponding glycosides

2,4,5

Leaf

- volatile oil (1.0-2.5%): 1,8-cineole (15-55%); camphor (5-25%); α-pinene (9-26%); camphene (2.5-12%); β-pinene (2-9%);

borneol (1.5-6%); limonene (1.5-5%); bornyl acetate (1-5%); isobutyl acetate; β-caryophyllene; p-cymene; linalool; myrcene; α-

terpineol (12-24%); verbenol

- diterpenes (up to 4.6%): carnosic acid; carnosol; isorosmanol; rosmadiol; rosmaridiphenol; rosmanol; rosmariquinone;

triacetylrosmanol; dimethylrosmanol

- triterpenes: oleanolic acid (10%); ursolic acid (2-5%); α-amyrin; β-amyrin; epi-α-amyrin; 19-α-ursolic acid; 2-β-hydroxy

oleanolic acid; betulin

- phenolic acids (2-3%): rosmarinic acid (3.5%); chlorogenic acid; neo-chlorogenic acid; caffeic acid; labiatic acid

- flavonoids: genkwanin; cirsimarin; diosmetin; apigenin; luteolin; nepetin; nepitrin; diosmin; hesperidin; homoplantiginin;

phegopolin

- alkaloids: rosmaricin; isorosmaricine

- tannins

- saponins

- glycolic acid and glyceric acid

- vitamin C; vitamin P

- choline

5,22,35,36,75

Leaf Oil

- α-pinene (8-25%), β-pinene (7.6%); eucalyptol (20-50%), camphor (10-27.6%), borneol (20%), 1,8-cineole (15.8%); β-myrcene

(10%); camphene (5.2-5.8%), limonene (5.9%); p-cymene (4.8%); β-caryophyllene (3.1%); verbenone (2.6%); linalool

35,40,41,73,76

- From one sample (concentration in the oil):

- monoterpenoid esters (24.76%): bornyl acetate (20.86%); linalyl acetate (2.90%); terpinyl acetate (1.0%)

- monoterpenoid alcohols (23.78%): borneol (8.25%); linalool (5%); isoborneol (4.13%); γ-terpineol (2.94%); α-terpineol

(1.9%); terpinene 4-ol (1.43%); carveol (0.13%)

- monoterpenoid ketones (18.67%): L-camphor (14.06%); verbenone (2.56%); carvone (1.9%); α-thujone (0.15%)

- monoterpenoid ethers (10.86%): methyl eugenol (5.46%); 1,8-cineole (5.05%); linalool oxide (0.35%)

- sesquiterpenes (8.96%): β-caryophellene (4.31%); caryophellene oxide (3.19%); spathulenol (1.27%); α-copene (0.19%)

- phenols (4.06%): thymole (3.06%); carvacrol (0.91%); methyl chavicol (0.19%)

- monoterpenes (3.4%): p-cymene (1.15%);

-pinene (0.95%); camphene (0.81%); myrcene (0.22%); limonene (0.15%)

Flower

- carnosic acid, carnosol, 12-O-methylcarnosic acid, at levels that are less than that found in the leaves

- highest levels of rosmarinic acid are found in the flower

- the flavones genkawanin and isoscutellarein 7-O-glucoside were not found in a DMSO extract

Seed

- 560.5 µg/g

-tocotrienol; 300.3 µg/g β-tocotrienol; 109.4 µg/g

-tocotrienol

Essential Oil

- mainly monoterpenes:

-pinene (20.1-21.7%),

-pinene; camphene; limonene; 1,8-cineole (23.5-26.5%); eucalyptol (4.5%);

and borneol

- camphor (7.2%); berbonone (7.6%); linalool; verbenol; terpineol; 3-octanone; isobornyl acetate

4,78,79

Table 5. Rosmarinus Officinalis (Rosemary) Leaf Extracts (CO

extract) – Certificates of Analysis

Analytical Detail

Specifications (%)

Results (%)

Rosmarinus Officinalis (Rosemary) Extract (CO

)

Essential Oil Content

78-88

Volatile components:

-pinene

8-12

11.4

camphene

n.s.

4.0

-pinene

n.s.

3.7

myrcene

n.s.

2.7

p-cymene

n.s.

1.2

limonene

2-4

2.4

1,8-cineole

>40

41.3

linalool

n.s.

0.83

camphor

6-13

13.0

borneol

n.s.

3.8

-terpineol

n.s.

3.9

verbenone

n.s.

0.45

bornyl acetate

n.s.

0.94

carophyllene

3-10

4.7

Rosmarinus Officinalis (Rosemary) Leaf Extract (CO

; 14% diterpene phenols) (and) Helianthus Annuus Seed Oil

Essential Oil Content

1.9

Phenolic diterpenes:

rosmanol

n.s.

0.07

7-methyl-rosmanol

n.s.

0.09

carnosol

n.s.

1.2

carnosolic acid

n.s.

10.5

12-methyl-carnosolic acid

n.s.

2.4

sum of phenolic diterpenes

13-15

14.3

Reference antioxidant compounds (carnesol +

carnosic acid, calculated as carnosic acid)

n.s.

9.5

Ursolic Acid

n.s,

0.43

Oleanolic Acid

n.s.

0.62

residual ethanol

0.71

water content

0.30

Rosmarinus Officinalis (Rosemary) Leaf Extract (CO

; 25% diterpene phenols) (and) Helianthus Annuus Seed Oil

Essential Oil Content

3.0

Phenolic diterpenes:

rosmanol

n.s.

0.13

7-methyl-rosmanol

n.s.

0.18

carnosol

n.s.

1.4

carnosolic acid

n.s.

18.7

12-methyl-carnosolic acid

n.s.

4.5

sum of phenolic diterpenes

24-26

24.9

Ursolic Acid

n.s.

0.29

Oleanolic Acid

n.s.

0.51

residual ethanol

0.39

water content

0.91

Rosmarinus Officinalis (Rosemary) Leaf Extract (CO

; 25% diterpene phenols) (and) Helianthus Annuus Seed Oil

12,15

Essential Oil Content

1.7

Phenolic diterpenes:

rosmanol

n.s.

0.13

7-methyl-rosmanol

n.s.

0.32

carnosol

n.s

2.9

carnosic acid

> 16

20.6

12-methyl-carnosic acid

n.s.

1.0

sum of phenolic diterpenes

24-26

25.0

Ursolic Acid

n.s.

0.42

Oleanolic Acid

n.s.

0.52

residual ethanol

0.33

water content

0.15

n.s. – not specified

Table 6. Differences in constituent profiles in Rosmarinus officinalis (rosemary) Leaf Extract based on extraction method

Extraction Method

Constituent (ppm) dried leaves

supercritical CO

acetone

ethanol extract,

partially deodorized

ethanol extract,

deodorized

decolorized and deodorized

using hexane and ethanol

Triterpenes

betulin

<4760

6000

5600

8450

9460

6790

amyrin

<500

200

160

230

360

oleanic+ursolic acid

148,100

48,500

100,500

119,800

164,500

60,000

Flavonoids

genkwanin

2.9

0.65

1.60

2.30

3.66

2.1

Volatiles

1,8-cineole

56,100

1700

1320

camphor

25,200

220

2360

2080

120

borneol

10,000

960

840

Heavy Metals

lead

2.90

0.09

0.03

0.13

0.15

0.18

arsenic

1.14

<0.034

0.05

0.25

0.32

* standardized to 10% carnosic acid + carnosol content

Table 7. Toxicity information on constituents of Rosmarinus officinalis (rosemary)

Component

Toxicity information

Phenol Acids

Caffeic Acid

- in a MMC-induced SCE assay in human lymphocytes, 100 μM caffeic acid enhanced MMC-induced SCEs by 55%; 100 μM

caffeic acid alone enhanced MMC-induced SCEs by 26%

- caffeic acid is reported to penetrate skin and have UV photoprotective activity

- humans and animals metabolize caffeic acid to the same metabolites, and hydrolyze chlorogenic acid to caffeic acid; IARC con-

cluded that there is sufficient evidence for carcinogenicity in animals of caffeic acid; no data were available on the carcinogenicity

in humans, and IARC concluded that caffeic acid is possibly carcinogenic to humans

- the carcinogenic potency of caffeic acid, estimated based on an average human intake of 1 mg/kg bw/day, was less than 1000 can-

cer cases per 1,000,000 individuals; in rats 1 or 2% (10,000 or 20,000 ppm) caffeic acid in the diet for 51 wks to 2 yrs induced pap-

illomas of the forestomach and renal adenomas; one study, in which rats were exposed to 2% (20,000 ppm) caffeic acid in the diet

for 2 yrs, showed treatment-induced carcinomas of the forestomach, whereas two studies with shorter exposure durations showed no

such effect; caffeic acid was shown to exert strong promotion activity for forestomach carcinogenesis; chronic exposure to caffeic

acid in the diet induced hyper

plasia of the forestomach (mice, rats, and hamsters), hyperplasia of the kidney (mice and rats), and in

creased liver and kidney wts (rats); few toxic effects resulted from acute exposure; subchronic dietary exposures did not induce clin-

ical symptoms of toxicity, however, hyperplasia of the forestomach was observed; some genotoxic effects seen in vitro but not in

vivo

Chlorogenic Acid

-an antioxidant that inhibited tumor promotion by phorbol esters in mice; some controversy exists over allergic reactions in green

coffee beans, but it was accepted that chlorogenic acid was not the allergen

-in mice, 2% (20,000 ppm) chlorogenic acid in the diet for 96 weeks induced papillomas and carcinomas of the forestomach,

alveolar type II-cell tumors of the lung, and renal cell adenomas; few toxic effects resulted from acute exposure; subchronic dietary

exposures did not induce clinical symptoms of toxicity, however, reduced kidney and adrenal wts and hyperplasia of the fore-

stomach were observed; some genotoxic effects seen in vitro but not in vivo

Flavonoids

epidemiological studies implicated high dietary intake levels of flavonoids in heart disease, but a study of cancer risk failed to find a

link; some evidence of genotoxicity in bacterial assays, but a European Organization of Cosmetic Ingredients Industries and

Services (UNITIS) report stated that flavonoids do not appear to be genotoxic to mammals in vivo; flavonoids are not considered

allergens

Diterpenes

Carnosic Acid

- is a known antioxidant;

in a toxicokinetic study in male Sprague-Dawley rats, carnosic acid was absorbed into the blood stream

after oral administration and was bioavailable, traces of the acid were found in the intestinal content, liver, and muscle tissue of the

abdomen and legs, carnosic acid was present in its free form, and the main route of elimination was the feces;

not mutagenic in an

Ames test, with or without metabolic activation, at doses equivalent to the concentration present in up to 6000 µg/plate of a

decolorized and deodorized rosemary leaf extract

Carnosol

- topical application of carnosol isolated from rosemary inhibited TPA-induced ear inflammation and tumor promotion in mice;

not mutagenic in an Ames test, with or without metabolic activation, at doses equivalent of the concentration present in up to 6000

µg/plate of a decolorized and deodorized rosemary leaf extract

Monoterpenes

these chemicals may be skin sensitizers

d- Limonene

- d-limonene consumption has been estimated as 0.2 -2 mg/kg bw/day; in men, oral intake induced transient proteinuria

- developmental toxicity in the form of delayed prenatal growth has been observed in mice, rats and rabbits exposed to d-limonene

during gestation, and skeletal anomalies have also been observed in the fetuses of exposed mice and rabbits;

- the few genotoxicity studies available indicated that d-limonene and its 1,2-epoxide metabolite are not genotoxic

- IARC found there are sufficient evidence for carcinogenicity in animals, concluding that d-limonene produces renal tubular

tumors

in male rats by a non-DNA-reactive mechanism, through an α

-globulin-associated response, and therefore, the mechanism by

which d-limonene increases the incidence of renal tubular tumors in male rats is not relevant to humans; no data were available on

the carcinogenicity in humans, and IARC concluded that d-limonene is not classifiable as to its carcinogenicity in humans

α-Pinene

negative in the Ames assay and a mouse micronucleus test

Table 7. Toxicity information on constituents of Rosmarinus officinalis (rosemary)

Component

Toxicity information

1,8-Cineole

positive in a sister chromatid exchange assay; negative in a chromosomal aberration assay; negative in an Ames test

β-Myrcene

has been reported to cause dermatitis and conjunctivitis in humans; in Wistar rats, the NOAEL for embryotoxicity was 0.5 g/kg

bw/day and the NOAEL for peri- and post-natal developmental toxicity was 0.25 g/kg bw/day; was not genotoxic in vitro in SCE

and chromosomal aberration assays in Chinese hamster cells or human lymphocytes, but it did induce a slight increase is SCE in

cultured hepatic tumor cells; was not genotoxic in vivo in rat bone marrow cells;

up to 1.0 g/kg bw was administered in corn oil,

by gavage, to rats and mice, and there was clear evidence of carcinogenic activity in male rats (increased incidences of renal tubule

neoplasms) and male mice (increased incidences of hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma), and

equivocal evidence in female rats (increased incidences of renal tubule adenoma) and female mice (marginally increased incidences

of hepatocellular adenoma and carcinoma)

Linalool

- safe at up to 4.3% (20% in consumer fragrance); listed as a fragrance allergen by the European Commission

- International Fragrance Association (IFRA) stated pure linalool is not a sensitizer, but hydroperoxides and other oxidation

products have shown sensitizing properties; one of the major oxidation products of linalool was isolated and identified as 7-

hydroperoxy-3,7-dimethyl-octa-1,5-diene-3-ol; in sensitization studies in guinea pigs, linalool of high purity gave no reactions,

while linalool that had been oxidized for 10 wks sensitized the animals; it was concluded that autoxidation of linalool is essential for

its sensitizing potential

α,β-Thujone

α,β-thujone was not mutagenic in the Ames test; in the micronucleus test, negative in male and positive in female mice; β-thujone:

some evidence of carcinogenicity in male rats – significant incidence of cancers of the preputial gland in male rats given 25 mg/kg

by gavage, and an increase in adrenal gland tumors in male rats may have been due to β-thujone; no increase in in cancer incidence

in female rats (dosed with up to 50 mg/kg by gavage) or male or female mice (dosed with up to 25 mg/kg by gavage); all rats dosed

with 50 mg/kg and all female mice dosed with 25 mg/kg died

Methyleugenol

- IARC concluded that there is sufficient evidence in experimental animals for carcinogenicity; no data were available on the

carcinogenicity in humans, and IARC concluded that methyleugenol is possibly carcinogenic to humans

- IFRA stated available metabolic, biochemical and toxicological data in laboratory species provide clear evidence of non-linearity

in the dose-response relationship with respect to metabolic activation and mechanisms associated with carcinogenic effects; con-

sideration of these data indicates an NOEL in the rat in the dose-range of 1-10 mg/kg bw/day; based on the lower end of the NOEL

and applying a 1000x safety factor for systemic effects a daily dose of 60 μg/day is supported; taking into account a dermal penetra-

tion factor of 40% leads to an acceptable dose of 150 μg/day

Terpene Alcohols

α-Terpineol

- oral LD50 in mice, 2830 mg/kg; 1000 mg/kg bw/day for 2 wks caused reduced body wt gains and an increase in serum cholesterol;

not mutagenic in an Ames test or mouse lymphoma assay; did not induce pulmonary tumors in mice given i.p. injections; a dermal

irritant in animals studies, but not a dermal irritant in a 4-h clinical study; not a sensitizer in guinea pigs; in clinical patch tests, 5%

in pet. had 1/1606 positive and 11/1606 questionable reactions in one study and 2/1200 positive reactions in another

Ursolic acid

topical application of ursolic acid isolated from rosemary inhibited TPA-induced ear inflammation and tumor promotion in mice

Triterpene Alcohols

hepatoprotective and anti-carcinogenic activity has been suggested for lupeol; no toxicity data were available; triterpene alcohols

were considered to have intermediate risk

Table 8. Frequency and concentration of use according to duration and type of exposure

# of Uses

Max. Conc. of Use (%)

# of Uses

Max. Conc. of Use (%)

# of Uses

Max. Conc. of Use (%)

Rosmarinus Officinalis (Rosemary)

Extract

Rosmarinus Officinalis (Rosemary)

Flower Extract

Rosmarinus Officinalis (Rosemary)

Flower/Leaf/Stem Extract

Totals*

404

0.00004-0.16

0.0024

Duration of Use

Leave-On

247

0.00096 – 0.051

0.0024

Rinse Off

154

0.00004 -0.16

Diluted for (Bath) Use

Exposure Type

Eye Area

0.01-0.05

Incidental Ingestion

0.011

Incidental Inhalation-Spray 93

; 64

0.00096-0.001

; 1

NR NR NR

Incidental Inhalation-Powder

0.05

Dermal Contact

306

0.00096-0.16

0.0024

Deodorant (underarm) NR

not spray: 0.0098

aerosol: 0.0098-0.012

NR NR NR not spray: 0.0024

Hair - Non-Coloring

116

0.00004-0.003

Hair-Coloring

Nail

Mucous Membrane

0.0005-0.16

Baby Products

Rosmarinus Officinalis (Rosemary)

Leaf

Rosmarinus Officinalis (Rosemary)

Leaf Extract

Rosmarinus Officinalis

(Rosemary) Leaf Oil

Totals*

0.002

729

0.00001-10

474

0.00001-1.5

Duration of Use

Leave-On

0.002

473

0.00001-10

308

0.0003-1.5

Rinse Off

254

0.00001-3

141

0.00001-0.12

Diluted for (Bath) Use

0.0002-0.04

0.5-0.97

Exposure Type

Eye Area

0.002-3

Incidental Ingestion

0.00001-0.009

0.008

Incidental

Inhalation-Spray 1

0.002

8; 185

;

110

0.001-0.5

aerosol: 0.0016

pump spray: 0.0001-0.005

0.00002-0.5

; 106

0.011-1.5

aerosol: 0.007

Incidental Inhalation

-Powder NR NR

; 110

0.0002

; 106

0.0003

Dermal Contact

460

0.00001-10

381

0.0003-1.5

Deodorant (underarm)

Hair - Non-Coloring

0.002

219

0.00001-0.5

0.00001-1.5

Hair-Coloring

0.04

Nail

0.005-0.053

Mucous Membrane

0.00001-3

0.0002-0.97

Baby Products

0.012

Rosmarinus Officinalis (Rosemary)

Leaf Powder

Rosmarinus Officinalis (Rosemary)

Leaf Water

Rosmarinus Officinalis

(Rosemary) Water

Totals*

0.05

0.000069-1

---

Duration of Use

Leave-On

0.000069-1

Rinse Off

0.05

0.00015-0.25

Diluted for (Bath) Use

Exposure Type

Eye Area

0.000069-0.00016

Incidental Ingestion

0.005

Incidental Inhalation-Spray

; 4

; 1

Incidental Inhalation-Powder

Dermal Contact

0.00009-0.36

Deodorant (underarm)

Hair - Non-Coloring

0.05

0.00019-1

Hair-Coloring

Nail

Mucous Membrane

0.005

Baby Products

Table 8. Frequency and concentration of use according to duration and type of exposure

# of Uses

Max. Conc. of Use (%)

# of Uses

Max. Conc. of Use (%)

# of Uses

Max. Conc. of Use (%)

Rosemary

Totals*

---

Duration of Use

Leave-On

---

Rinse Off

---

Diluted for (Bath) Use

---

Exposure Type

Eye Area

---

Incidental Ingestion

---

Incidental Inhalation-Spray

1; 2

---

Incidental Inhalation-Powder

1; 2

---

Dermal Contact

---

Deodorant (underarm)

---

Hair - Non-Coloring

---

Hair-Coloring

---

Nail

---

Mucous Membrane

---

Baby Products

---

* Because each ingredient may be used in cosmetics with multiple exposure types, the sum of all exposure types may not equal the sum of total uses

NR – not reported

Includes products that can be sprays, but it is not known whether the reported uses are sprays

Includes products that can be powders, but it is not known whether the reported uses are powders

Not specified whether a spray or a powder, but it is possible the use can be as a spray or a powder, therefore the information is captured in both categories

Plant part and type of preparation not known

Table 9. Single-dose toxicity studies

Test Article

Extraction

Solvent/Method

Species

No./Group

Vehicle

Conc/Dose Range

/Results

Reference

DERMAL

Rosmarinus Officinalis

(Rosemary) Leaf Oil

-----

rabbits

not stated

>10 ml/kg

Rosmarinus Officinalis

(Rosemary) Leaf Oil

-----

rabbits

not stated

>10 g/kg

ORAL

Rosmarinus Officinalis

(Rosemary) Leaves – 2

samples; one harvested in

autumn (112.7, 477.8,

700.1 µg/mg extract car-

nosol, carnosic acid, total

diterpenes, respectively)

and one in spring (45.9,

245.9, 343.1 µg/mg ex

tract carnosol, carnosic

acid, total diterpenes,

respectively)

supercritical CO

Wistar rats

6 M/6F

corn oil

2 g/kg bw

8,22,40-

(gavage)

>2 g/kg

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(see Table 5 for

composition)

ethanol extract,

partially

deodorized

mice

not stated

none stated

8.5 g/kg bw (males)

10 g/kg bw (females)

>8.5 g/kg bw (males)

>10 g/kg bw (females)

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(see Table 5 for

composition)

ethanol extract,

deodorized

mice

not stated

none stated

24 g/kg bw (males)

28.5 g/kg bw

(females)

>24 g/kg bw (males)

>28.5 g/kg bw

(females)

Rosmarinus Officinalis

(Rosemary) Leaf Oil (see

Table 4 for composition)

hydrodistillation

Swiss

albino rats

20/group

-----

2-9 g/kg bw (gavage)

= 5.50 g/kg bw

= 1.10 g/kg bw

100

= 9 g/kg bw

Rosmarinus Officinalis

(Rosemary) Leaf Oil (see

Table 5 for composition)

-----

rats

not stated

none stated

not stated

5 ml/kg bw

Table 10. Repeated-Dose Toxicity Studies

Test Article

Extraction

Solvent/Method

Animals/Group

Study Duration

Vehicle

Dose/Concentration

Parameters Examined

Results*

Reference

ORAL

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

ethanol extract,

partially deodorized

mice; no./group

not stated

5 days (gavage)

none stated

4300 mg/kg bw (males)

5000 mg/kg bw (females)

parameters included signs of

toxicity, body wts, feed con-

sumption, organ wts, gross

lesions

- no mortality

- body wt increased slightly in males, but no

changes were seen in females; “marked increase” in

fatty liver was observed in males after repeated

administration

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table for composition)

ethanol extract,

deodorized

mice; no./group

not stated

5 days (gavage)

none stated

11,800 mg/kg bw (males)

14,100 mg/kg bw (females)

-parameters as above

- no changes in body wts; liver wts of females were

slightly increased; fatty livers were observed in test

animals at necropsy.

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

acetone

rats; no./group not

stated

14 day (diet)

-----

up to 3800 mg/kg diet

- parameters included signs

of toxicity, body wts, feed

consumption, clinical chem

- no treatment-related signs of toxicity, mortality, or

changes in body wts or feed consumption

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

supercritical CO

rats; no./group not

stated

14 days (diet)

-----

up to 2400 mg/kg diet

- parameters as above

- no treatment-related signs of toxicity, mortality, or

changes in body wts or feed consumption

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

acetone

20 rats/group

13 wks (diet)

-----

300, 600, 2400, or 3800

mg/kg diet

- parameters included signs

of toxicity, body wts, feed

consumption, gross and

microscopic lesions, clinical

chem, hematology, organ wts

- variations in clinical chemistry parameters at

times were stat sig, but the researchers stated that

because the changes were inconsistent, they were

not considered dose-related

- stat. sig, decrease in alkaline phosphate in the

3800 mg/kg group

- NOAEL was 3800 mg/kg diet

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

supercritical CO

20 rats/group

13 wks (diet)

-----

300, 600, or 2400 mg/kg diet

- parameters as above

- variations in clinical chemistry parameters at

times were stat sig; the researchers stated that

because the changes were inconsistent, they were

not considered dose-related

- a marginal reduction in body weights and feed

consumption in the animals of the 2400 mg/kg diet

groups were attributed to a lack of palatability of

the feed

- changes were more notable in females

- NOAEL was 2400 mg/kg diet (equiv. to 180 and

200 mg/kg bw/day for males and females,

respectively)

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

supercritical CO

female rats; no./

group not stated

91 days (diet);

28-day recovery

period

-----

0 or 2400 mg/kg diet (equiv.

to 0 or 195 mg/kg bw/day)

parameters included signs of

toxicity, body wts, feed con-

sumption, gross and micro-

scopic lesions, organ wts

- slight increase in liver wts after 91-days of dosing,

but not in those killed after the 28-day recovery

period

- an increase in microsomal protein concentration

observed after 91 days of dosing was also reversible

- no notable effects on the activity of the liver

enzymes CYP1A, CYP2B, or CYP3A

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

ethanol extract,

partially deodorized

Sprague-Dawley

rats; no./group not

stated

90 days (diet)

-----

0, 500, 1500,or 5000 mg/ kg

diet (equiv. to 0, 40, 120, or

400 mg/kg bw/day)

parameters included signs of

toxicity, body wts, feed con-

sumption, microscopic le-

sions, organ wts, hematology

- body wts in the high dose group were very slightly

reduced, most likely as a result of decreased feed

consumption

- a dose-response relationship was observed for

relative liver-to-body wt, in which a a slight but stat

sig increase was observed

- no microscopic changes in the liver were reported

Table 10. Repeated-Dose Toxicity Studies

Test Article

Extraction

Solvent/Method

Animals/Group

Study Duration

Vehicle

Dose/Concentration

Parameters Examined

Results*

Reference

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

ethanol extract,

deodorized

Sprague-Dawley

rats; no./group not

stated

90 days (diet)

-----

0, 500, 1500,or 5000 mg/ kg

diet (equiv. to 0, 40, 120, or

400 mg/kg bw/day)

-parameters as above

- a dose-response relationship was observed for

relative liver-to-body wt; extracts; a slight but stat

sig increase was observed

- no microscopic changes in the liver were reported

Rosmarinus Officinalis

(Rosemary) Leaf Extract (see

Table 5 for composition)

hexane and ethanol

(2-step extraction)

Sprague-Dawley

rats; no./group not

stated

3 mos (diet); 28-

day interim

group; 1-mo

recovery period

-----

0, 1000, 2500, or 5000 mg/

kg diet (equiv. to 0, 65, 164,

or 320 mg/kg bw/day)

- parameters as above

- no signs of toxicity, no mortality and no gross

lesions at necropsy

- reversible dose-dependent increases in absolute

liver wts and relative liver-to-body wts; stat sig in

the high dose group only

- treatment-related increase in bile duct hyperplasia

at the interim necropsy; the incidence was decreased

at the end of dosing and not seen after recovery

- in females, a decrease in pancreas wt was

observed at the interim necropsy

- no stat sig changes in hematology parameters, and

no microscopic changes

- the NOAEL was at least 320 mg/kg bw/day

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(after the volatile oil [1.1%]

was removed)

absolute ethanol

Swiss albino mice;

6M/group

3 wks (gavage)

olive oil

1500 mg/kg extract

controls – olive oil

no stat sig changes in relative liver, spleen, heart, or

lung wt to body wt compared to controls; there were

no stat sig changes in clinical chemistry parameters

single dose CCl

(gavage), then 3

wks extract

(gavage)

olive oil

3.3% CCl

(100 mg/kg bw)

1500 mg/kg extract

- with CCl

only, stat sig increases in relative liver

to body wt (18%) and spleen to body wt (45.6%)

compared to olive oil controls; CCl

affected all

measured clinical chemistry parameters

- with the extract, the increase in relative spleen to

body wt was stat sig, but not as great as with CCl

alone (34.9%); there was no stat sig increase in

relative liver to body wt; many of the changes in

clinical chemistry values were reduced or were non-

stat sig

Rosmarinus Officinalis

(Rosemary) Leaf Oil (see

Table 4 for composition)

hydrodistillation

Swiss albino mice;

6M/group

3 wks (gavage)

-----

1100 mg/kg bw

controls – olive oil

no stat sig changes in relative liver, spleen, heart, or

lung wt to body wt compared to controls; there were

no stat sig changes in clinical chemistry parameters

single dose CCl

(gavage), then 3

wks oil (gavage)

olive oil

(for CCl

)

3.3% CCl

(100 mg/kg bw)

1100 mg/kg extract

- (effects of CCl

only are described above)

- with the oil, the increases in relative liver to body

wt (9.8%) and spleen to body wt (38.8%) were stat

sig, but not as great as with CCl

alone;

many of the

changes in clinical chemistry values were reduced

but were still stat sig

*if not described in the results, details on histopathology or organ weights were not provided

Abbreviations: CCl

: - carbon tetrachloride; conc – concentration; equiv. – equivalent; NOAEL – no-observable adverse effect level; stat sig – statistically significant

Table 11. Genotoxicity studies

Test Article

Extraction

Solvent/Method

Conc./Vehicle

Procedure

Test System

Results

Reference

IN VITRO

Rosemary Extract (not de-

fined; water-soluble; con-

tained 17% rosmarinic

acid)

-----

50, 100, or 200 µg/

plate

Ames test, with and without

metabolic activation

S. typhimurium TA98

not mutagenic

as above

-----

50 µg/ml (highest non-

cytotoxic dose)

comet assay

human hepatoma cell line

(HepG2)

not genotoxic

Rosemary Extract (not de-

fined; oil-soluble; con-

tained 50.27% carnosic

acid and 5.65% carnosol)

-----

50, 100, or 200 µg/

plate

Ames test, with and without

metabolic activation

S. typhimurium TA98

not mutagenic

as above

-----

5 µg/ml (highest non-

cytotoxic dose)

comet assay

human hepatoma cell line

(HepG2)

not genotoxic

Rosmarinus Officinalis

(Rosemary) Leaf Extract

supercritical CO

up to 5000 µg/plate

bacterial assay, with and without

metabolic activation

S. typhimurium TA98, TA100,

TA1535, TA1537, TA102

not mutagenic

- in TA102 only, toxicity at the highest dose

with metabolic activation

Rosmarinus Officinalis

(Rosemary) Leaf Extract

ethanol extract,,

partially deodorized

up to 20,000 µg/plate

bacterial assay, with and without

metabolic activation

S. typhimurium TA98, TA100,

TA1535, TA1537, TA102

not mutagenic

- some bactericidal effects in all strains; ef-

fects were reduced with metabolic activation

Rosmarinus Officinalis

(Rosemary) Leaf Extract

ethanol extract,

deodorized

up to 20,000 µg/plate

bacterial assay, with and without

metabolic activation

S. typhimurium TA98, TA100,

TA1535, TA1537, TA102

not mutagenic

- some bactericidal effects in all strains; ef-

fects were reduced with metabolic activation

Rosmarinus Officinalis

(Rosemary) Leaf Extract

hexane and ethanol

(2-step extraction)

up to 6000 µg/plate

Ames test, with and without meta-

bolic activation

S. typhimurium TA97, TA98,

TA100, TA102

-mutagenic in TA102 in one set of trials; not

reproducible with less cytotoxic conc

-not mutagenic in the other strains

- without metabolic activation: bactericidal

for all strains at 3000-6000 µg/plate; bacteri-

cidal to TA102 at almost all dose levels

-with metabolic activation, bactericidal only

at the highest dose level, if at all

Rosmarinus Officinalis

(Rosemary) Leaf Extract

ethanol extract,

partially deodorized

up to 100 mg/ml

chromosomal aberration assay, with

and without metabolic activation

human lymphocytes

not genotoxic

Rosmarinus Officinalis

(Rosemary) Leaf Extract

hexane and ethanol

(2-step extraction)

not clearly specified

but at least up to 50

µg/ml without and 35

µg/ml with metabolic

activation

gene-locus mutation assay, with

and without metabolic activation

thymidine kinase (tk) and

hgprt loci of a human lympho-

blastoid cell line (TK6)

-not genotoxic without metabolic activation

at up to 50 µg/ml

- 35 µg/ml increased mutations in the tk, but

not the hgprt, locus with activation; the in-

crease was stat sig when compared to solvent

control, but not when compared to untreated

cells; determined to be not mutagenic under

the conditions used because of a lack of a

dose-dependent increase in mutation fre-

quency and a lack of a stat sig increase of

mutation frequency compared to controls

Rosmarinus Officinalis

(Rosemary) Leaf Oil

-----

not stated

Ames test

not stated

negative

Table 11. Genotoxicity studies

Test Article

Extraction

Solvent/Method

Conc./Vehicle

Procedure

Test System

Results

Reference

IN VIVO

Rosmarinus Officinalis

hydro-alcoholic

6.43, 100, and 200

mg/kg bw

chromosomal aberration assay

Wistar rats; 6/group

not genotoxic

Rosmarinus Officinalis

hydro-alcoholic

6.43, 100, and 200

mg/kg bw

micronucleus assay

Wistar rats; 6/group

not genotoxic

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(after the volatile oil

[1.1%] was removed)

absolute ethanol

1500 mg/kg bw/day in

olive oil

micronucleus test; dosed by gavage

for 7 days; negative controls were

given olive oil; positive controls

were given a single i.p. dose of 100

mg/kg bw CPA; bone marrow cells

collected 24 h after dosing

Swiss albino mice

not genotoxic; no stat sig change in the num-

ber of MNPCE or NCE or in PCE/NCE

Rosmarinus Officinalis

(Rosemary) Leaf Oil (see

Table 4 for composition)

hydrodistillation

1100 mg/kg bw/day

same protocol

Swiss albino mice

no stat sig change in no. of MNPCE

no. of NCE was stat sig decreased (p<0.05)

PCE/NCE was stat sig increased (p<0.01)

Rosmarinus Officinalis

(Rosemary) Leaf Oil

hydrodistillation

300, 1000, or 2000

mg/kg bw (by gavage)

chromosome aberration assay;

single 0.5 ml dose; negative

controls were given distilled water;

positive controls were dosed with

50 mg CPA/kg; bone marrow cells

collected 24 h after dosing

Wistar rats; 3M/3F per group

- chromosomal aberrations without gaps

were stat sig increased at 2000 mg/kg bw

- mitotic index was stat sig increased with

300 mg/kg, but not with other doses or the

positive control

Rosmarinus Officinalis

(Rosemary) Leaf Oil

hydrodistillation

300, 1000, or 2000

mg/kg bw (by gavage)

micronucleus test; single 0.5 ml

dose; negative controls were given

distilled water; positive controls

were dosed with 50 mg CPA/kg;

bone marrow cells collected 24 h

after dosing

Swiss mice; 3M/3F per group

- stat sig increase in MNPCEs with 1000 and

2000 mg/kg bw

- PCE/NCE was not stat sig different from

controls

Rosmarinus Officinalis

(Rosemary) Leaf Oil

hydrodistillation

300, 1000, or 2000

mg/kg bw (by gavage)

micronucleus test; protocol as

above; bone marrow cells collected

24 h after dosing

Wistar rats; 3M/3F per group

stat sig increase in MNPCEs with 2000

mg/kg bw

Rosmarinus Officinalis

(Rosemary) Leaf Oil

hydrodistillation

300, 1000, or 2000

mg/kg bw (by gavage)

comet assay; single 0.5 ml dose;

negative controls were given dis-

tilled water; positive controls were

dosed with 50 mg CPA/kg; liver

and peripheral blood cells collected

24 h after dosing

Swiss mice; 3M/3F per group

all 3 doses induced stat sig increases in DNA

damage in peripheral blood cells and liver

cells; most of the damaged cells showed

minor damage, very few had a large amount

of damage

mixture containing 19%

Rosmarinus officinalis

(rosemary) leaves, 71..5%

St. John’s Wort; 9.5%

spirulina

-----

0, 380, 760, or 1520

mg/ kg bw/day in

water (gavage)

micronucleus test; mice were dosed

for 7 days; femoral bone marrow

cells were used

male Swiss albino mice;

30/group

- stat. sig. increase in MNPCEs with 760 and

1520 mg/kg bw/day

- PCE/NCE was not stat sig different from

controls

mixture defined above

-----

0, 380, 760, or 1520

mg/ kg bw/day in

water (gavage)

chromosomal aberration assay;

mice were dosed for 7 days and

killed 19 days after last dose

male Swiss albino mice;

30/group

- stat sig increased in frequency of aneu-

ploidy with 760 and 1520 mg/kg bw/day

- % polyploids and total % aberrations were

stat sig increased at these doses

Table 11. Genotoxicity studies

Test Article

Extraction

Solvent/Method

Conc./Vehicle

Procedure

Test System

Results

Reference

mixture defined above

-----

0, 380, 760, or 1520

mg/ kg bw/day in

water (gavage)

assay for spermatozoa abnormality;

mice were dosed for 7 days and

killed 5 wks after last dose

male Swiss albino mice;

30/group

- stat sig increase in frequency of banana-

shaped, swollen achrosome, and triangular

head sperm abnormalities with 1520 mg/kg

bw/day

- % total spermatozoa abnormalities stat sig

increased with 1520 mg/kg bw/day

ANTI-MUTAGENIC EFFECTS

IN VITRO

Rosemary Extract (not

defined; contained 8.8-

10.6% carnosic acid and

1.2-1.4% carnosol) +

tBOOH

-----

≤0.8 mg/ml in medi-

um-

chain triglycerides;

only the carnosic acid

and carnosol were

soluble

Ames test; 0.5 ml rosemary extract

was incubated with 0.5 ml tBOOH

S. typhimurium TA102

stat sig reduced tBOOH-induced

mutagenicity

Rosemary Extract (not de-

fined; water-soluble; con-

tained 17% rosmarinic

acid) + IQ

-----

50, 100, or 200 µg/

plate extract

10 ng/plate IQ

Ames test, with metabolic

activation

S. typhimurium TA98

a stat sig reduction in IQ-induced genotoxi-

city was observed only at the highest dose

as above + NQNO

-----

0, 50, 100, or 200 µg/

plate extract

500 ng/plate NQNO

Ames test, without metabolic

activation

S. typhimurium TA98

no stat sig effect on NQNO-induced

genotoxicity

as above + tBOOH

-----

0, 0.05, 0.5, 5, or 50

µg/ml extract; 0.05

mM tBOOH

Comet assay; pretreatment with

extract for 21 h, followed by 20

min exposure to tBOOH

human hepatoma cell line

(HepG2)

stat sig reduction in tBOOH-induced DNA

damage at all doses; the reduction was not

dose-dependent – 0.05 µg/ml caused a

greater reduction than 0.5 µg/ml

as above + tBOOH

-----

0, 0.05, 0.5, 5, or 50

µg/ml extract; 0.05

mM tBOOH

Comet assay; co-treatment with

extract and tBOOH for 20 min

human hepatoma cell line

(HepG2)

no stat sig effect on tBOOH-induced DNA

damage

as above + tBOOH

-----

0, 0.05, 0.5, 5, or 50

µg/ml extract; 0.05

mM tBOOH

Comet assay; pretreatment with

extract for 21 h, followed by co-

treatment with extract and tBOOH

for 20 min

human hepatoma cell line

(HepG2)

stat sig reduction in tBOOH-induced DNA

damage at all except the lowest dose

as above + BaP

-----

0, 0.05, 0.5, 5, or 50

µg/ml extract; 40 µM

BaP

by co-treatment with extract and

BaP for 21 h

human hepatoma cell line

(HepG2)

stat sig reduction in BaP-induced DNA

damage only at the highest dose

as above + PhIP

-----

0, 0.05, 0.5, 5, or 50

µg/ml extract; 80 µM

PhIP

Comet assay; by co-treatment with

extract and PhIP for 21 h

human hepatoma cell line

(HepG2)

stat sig reduction in PhIP-induced DNA

damage only at the highest dose

Rosemary Extract (not de-

fined; oil-soluble; con-

tained 50.27% carnosic

acid and 5.65% carnosol)

+ IQ

-----

50, 100, or 200 µg/

plate extract

10 ng/plate IQ

Ames test, with metabolic

activation

S. typhimurium TA98

suppressed IQ-induced mutations in a stat

sig, dose-dependent, manner

as above + NQNO

-----

50, 100, or 200 µg/

plate extract

500 ng/plate NQNO

Ames test, without metabolic

activation

S. typhimurium TA98

suppressed NQNO-induced mutations in a

stat sig, dose-dependent, manner

as above + tBOOH

-----

0, 0.05, 0.5, or 5 µg/

ml extract; 0.05 mM

tBOOH

comet assay; pretreatment with

extract for 21 h, followed by 20

min exposure to tBOOH

human hepatoma cell line

(HepG2)

stat sig reduction in tBOOH-induced DNA

damage at all doses

Table 11. Genotoxicity studies

Test Article

Extraction

Solvent/Method

Conc./Vehicle

Procedure

Test System

Results

Reference

as above + tBOOH

-----

0, 0.05, 0.5, or 5 µg/

ml extract; 0.05 mM

tBOOH

comet assay; co-treatment with

extract and tBOOH for 20 min

human hepatoma cell line

(HepG2)

no stat sig effect on tBOOH-induced DNA

damage

as above + tBOOH

-----

0, 0.05, 0.5, or 5 µg/

ml extract; 0.05 mM

tBOOH

comet assay; pretreatment with

extract for 21 h, followed by co-

treatment with extract and tBOOH

for 20 min

human hepatoma cell line

(HepG2)

stat sig reduction in tBOOH-induced DNA

damage at all doses; the reduction was not

dose-dependent`

as above + BaP

-----

0, 0.05, 0.5, or 5 µg/

ml extract; 40 µM BaP

by co-treatment with extract and

BaP for 21 h

human hepatoma cell line

(HepG2)

stat sig reduction in BaP-induced DNA

damage at the two highest doses

as above + PhIP

-----

0, 0.05, 0.5, or 5 µg/

ml extract; 80 µM

PhIP

by co-treatment with extract and

PhIP for 21 h

human hepatoma cell line

(HepG2)

stat sig reduction in PhIP-induced DNA

damage at the two highest doses

IN VIVO

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(after the volatile oil

[1.1%] was removed) +

CPA

absolute ethanol

1500 mg/kg bw/day in

olive oil

micronucleus test; dosed by gavage

with the extract for 7 days, then

given a single i.p. dose of 100

mg/kg bw CPA; bone marrow cells

collected 24 h after dosing; olive

oil was used as a negative control

Swiss albino mice

stat sig increase in the number of MNPCE

and NCE compared to olive oil only; no stat

sig change in PCE/NCE

Rosmarinus Officinalis

(Rosemary) Leaf Oil (con-

tained 20.86% bornyl ace-

tate; 16.24% L-camphor,

and 8.25% borneol) + CPA

hydrodistillation

1100 mg/kg bw/day

micronucleus test; dosed by gavage

with the oil for 7 days, then given a

single i.p. dose of 100 mg/kg bw

CPA; bone marrow cells collected

24 h after dosing; olive oil was

used as a negative control

Swiss albino mice

stat sig increase in the number of MNPCE

and NCE, and a stat sig decrease in

PCE/NCE, compared to olive oil only

Abbreviations: BaP – benzo(a)pyrene; conc – concentration; CPA - cyclophosphamide: IQ – 2-amino-3-methyl-3H-imidao[4,5-F]quinoline; MMS – methyl methanesulfonate; MNPCE – micronucleated

polychromatic erythrocytes; NCE – normochromatic erythrocytes; NQNO – 4-nitroquinoline-N-oxide; PCE/NCE – ratio of polychromatic erythrocytes to normochromatic erythrocytes; PhIP – 2-amino-1-

methyl-6-phenylimidazo[4,5-b]pyridine; stat sig – statistically significant; tBOOH - t-butyl hydroperoxide

Table 12. Effects on Tumor Promotion

Test Article

Extraction

Solvent/Method

Dose/Exposure

Route

Species

No./Group

Tumor Type

Carcinogenicity Model

Results

Reference

Rosmarinus Officinalis

(Rosemary) Leaf Extract

(contained 16.5-19.2% uro-

solic acid; 3.8-4.6% carno-

sol; 0.1-0.5% carnosic acid;

trace-0.1% miltirone)

methanol

1.2 or 3.6 mg;

dermal

CD-1 mice;

30F/grp

skin

- initiation: topical treatment with 200 nmol

DMBA in 200 µl acetone

- promotion: after 1 wk, topical treatment with

200 µl acetone (controls), 5 nmol TPA in 200 µl

acetone (carc grp), or 5 nmol TPA and extract in

200 µl acetone (RE grp), 2x/wk, for 20 wks

1.2 mg: decreased tumor/mouse by 48, 27,

and 28% after 7, 11, and 15 wks TPA

promotion

3.6 mg: decreased tumor/mouse by 84, 37,

and 48% after 7, 11, and 15 wks TPA

promotion

as above

methanol

1.2 or 3.6 mg; 5

min prior to

B(a)P; dermal

CD-1 mice;

30F/grp

skin

- initiation: topical treatment with 200 µl acetone

(controls) or with extract in 200 µl acetone (RE

grp) 5 min prior to each 20 nmol application of

B(a)P or 2 nmol DMBA, 1x/wk, for 10 wks

- promotion: after 1 wk, promotion with 15 nmol

TPA in 200 µl acetone, 2x/wk, for 20 wks

1.2 mg: decreased tumor/mouse by 15, 42,

and 54% after 9, 13, or 21 wks TPA

promotion

3.6 mg: decreased tumor/mouse by 62, 63,

and 64% after 9, 13, or 21 wks TPA

promotion

as above

methanol

3.6 mg; dermal

CD-1 mice;

30F/grp

skin

- initiation: topical treatment with 200 µl acetone

(controls) or 3.6 mg extract in 200 µl acetone

(RE grp) at 120, 60, and 5 min before topical

application of 200 nmol B(a)P in 200 µl acetone

- promotion: after 1 wk, 15 nmol in 200 µl ace-

tone, 2x/wk, for 20 wks

decreased tumor/mouse by 83, 81, and

58% after 9, 13, or 21 wks TPA

promotion

Rosmarinus Officinalis

(Rosemary) Leaf Extract

DDW

500 mg/kg bw;

gavage

Swiss albino

mice; 12M/grp

skin

DMBA-initiated and croton oil-promoted skin

tumorigenesis

Grp 1: controls – topical treatment with 100 µl

acetone; DDW by gavage for 15 wks

Grp 2: 500 mg/kg bw/day RE in 100 µl DDW for

15 wks

Grp 3: single topical dose 100 µg DMBA in 100

µl acetone; 2 wks later, 1% croton oil in acetone,

3 x/wk; also, 100 µl by gavage for 15 wks

Grp 4: single topical dose 100 µg DMBA in 100

µl acetone; 500 mg/kg bw RE by gavage 7 days

before, during, and 7 days after DMBA; 2 wks

after DMBA, 1% croton oil in acetone, 3x/wk

Grp 5: single topical dose 100 µg DMBA in 100

µl acetone; after 2 wks, 500 mg/kg bw RE ex-

tract by gavage for 15 days and 1% croton oil in

acetone 3x/wk

Grp 6: single topical dose 100 µg DMBA in 100

µl acetone; 500 mg/kg bw RE by gavage 7 days

before DMBA until study end; 2 wks after

DMBA, 1% croton oil in acetone, 3x/wk

-a stat sig decrease in tumor number, di-

ameter, and weight and a stat sig increase

in the avg. latency period

was observed in

grps given RE compared to Grp 3 (the

carcinogen-control grp)

- blood serum and liver lipid peroxidation

level was stat sig de

creased in all RE grps

compared to grp 3

- Grp 6 had the greatest changes for all

the above parameters

- no tumors were found in animals given

RE only

- RE had no effect on body weight gains

Rosmarinus Officinalis

(Rosemary) Leaf Extract

DDW

1000 mg/kg bw

in DDW; gavage

Swiss albino

mice; 12M/grp

skin

DMBA-initiated and croton oil-promoted skin

tumorigenesis

-same protocol as above (Grps 1-6), except 1000

mg/kg bw RE was used

- stat sig decrease in tumor burden and

tumor yield, and a stat sig

increase in avg.

latency period, in grps given RE com-

pared to Grp 3 (the carcinogen-control

grp); tumor incidence was decreased

- blood serum lipid peroxidation level was

stat sig decreased in all RE grps, and the

liver glutathione levels stat sig increased,

compared to grp 3

- RE did not cause any adverse effects; no

tumors were seen in the RE-only grp.

Table 12. Effects on Tumor Promotion

Test Article

Extraction

Solvent/Method

Dose/Exposure

Route

Species

No./Group

Tumor Type

Carcinogenicity Model

Results

Reference

Rosmarinus Officinalis

(Rosemary) Extract

not specified

1.0%, in diet

Sprague-

Dawley rats;

20F/grp

mammary

- rats were fed untreated or RE-supplemented

diet throughout the study (16 wks post-DMBA)

- after 27 days of the test diet, each rat was dosed

with 30.9 mg/kg bw DMBA in corn oil by

gavage

- the incidence of palpable mammary

tumors was less in the RE-fed rats than

the controls; at study termination, the

tumor incidence was 47% less; this differ-

ence was stat sig

- the difference in tumors per tumor-bear-

ing rat was not stat sig btwn the two grps

- at study termination, 94% and 90% of

tumor-bearing rats of the control and RE

groups, respectively, possessed mammary

adenocarcinomas

- RE had no effect on body wt

Abbreviations: B(a)P – benzo[a]pyrene; DDW – double-distilled water; DMBA – 7,12-dimethylbenz[a]anthracene; grp – group; GR – glutathione reductase; GSH – reduced glutathione; GST –

glutathione-s-transferase; RE – Rosmarinus officinalis (rosemary) leaf extract; stat sig – statistically significant; TPA – 12-O-tetradecanoylphorbol-13-acetate

Table 13. Dermal Irritations and Sensitization

Test Article

Concentration/Dose

Test Population

Procedure

Results

Reference

NON-HUMAN

4.4% rosmarinus officinalis

(rosemary) leaf oil (and

other essential oils)

tested at concentrations

up to 40%

Lewis rats

ointment was applied to the shaved skin of Lewis rats

twice daily, for 14 days

not irritating

no gross or microscopic lesions were

reported in the skin

rosmarinus officinalis

(rosemary) leaf oil

undiluted

rabbits

applications were made to intact and abraded rabbit

skin under occlusion; no other details were provided

moderately irritating

HUMAN

Rosmarinus officinalis

(rosemary) leaves

undiluted in sufficient

petrolatum for binding

234 patients with

contact dermati-

tis or eczema

patch test

21 had +/- reactions; 18 had a + reaction; 5

had a ++ reaction; no subjects had a +++

reaction

Rosmarinus officinalis

(rosemary) leaves extracted

with supercritical CO

undiluted in petrolatum

20 subjects

epicutaneous test using Finn chambers

weak irritant

1 positive reaction

Rosmarinus officinalis

(rosemary) leaves as an

absolute (soluble in hexane)

undiluted in petrolatum

25 subjects

epicutaneous test using Finn chambers

weak irritant

2 positive reactions

2% and 10%

23 subjects pre-

viously sensi-

tized to peru

balsam and/or

perfumes or fra-

grance materials

epicutaneous test using Finn chambers

weak effect

Table 13. Dermal Irritations and Sensitization

Test Article

Concentration/Dose

Test Population

Procedure

Results

Reference

Rosmarinus officinalis

(rosemary) leaves as a

concrete (insoluble waxes)

extracted in hexane

undiluted in petrolatum

20 subjects

epicutaneous test using Finn chambers

no reactions

2% and 10%

23 subjects pre-

viously sensi-

tized to peru

balsam and/or

perfumes or fra-

grance materials

epicutaneous test using Finn chambers

weak effect

cream containing 0.2%

rosmarinus officinalis

(rosemary) leaf extract

undiluted

20 subjects

a 24 h single insult occlusive patch test

not an irritant

no reactions were observed, and the

primary irritation index was 0.00

hair spray containing

0.0013% rosmarinus

officinalis (rosemary) leaf

extract

neat

102 subjects

modified Draize HRIPT

induction: occlusive patches were applied for 24 h, and

the sites were scored prior to the application of the

next patch; patches were applied 3x/wk for 3 wks; the

material was allowed to volatilize for 30 min prior to

application

challenge: after a 2-wk non-treatment period, chal-

lenge patches were applied to a previously untreated

site; the test sites were scored 24 and 72 h after

application

not an irritant or sensitizer

transient, barely perceptible to mild

responses were observed in some subjects,

but was not considered related to skin

irritation or an allergic reaction

sunscreen cream containing

0.2% rosmarinus officinalis

(rosemary) leaf extract

neat

27 subjects

maximization test

induction: an occlusive patch containing 0.1 ml of

0.25% aq. SLS was applied to the upper outer arm,

volar forearm, or back of each subject for 24 h; the

SLS patch was removed and an occlusive patch with

0.1 ml test material then applied for 48 or 72 h; the

patch was then removed and the test site examined; a

total of five SLS/test material patches were applied

during induction

challenge: after a 10-day non-treatment period, an

occlusive patch with 0.1 ml of a 5% aq. SLS solution

was applied to a previously untreated site for 1 h; this

patch was removed and an occlusive patch containing

0.1 ml undiluted test material was then applied for 48

h; the challenge site was graded 1 and 24 h after patch

removal

not a contact-sensitizer

no reactions were observed

Rosmarinus officinalis

(rosemary) leaf oil

10% in petrolatum

not specified

48-h closed patch test; details not provided

not an irritant

Rosmarinus officinalis

(rosemary) leaf oil

10% in petrolatum

25 subjects

maximization test; details not provided

not a sensitizer

Table 13. Dermal Irritations and Sensitization

Test Article

Concentration/Dose

Test Population

Procedure

Results

Reference

leave-on massage oil con-

taining 1.5% rosmarinus

officinalis (rosemary) leaf

oil

neat

104 subjects

HRIPT

induction: an occlusive patch containing 50 µl of un-

diluted test material was applied for 48 h; the patches

were then removed and a new patch applied; 9 induc-

tion patches were applied.

challenge: performed 12-14 days after induction at the

original test site and a previously untested site for 48

h; sites were scored at 48 and 96 h

patches of 0.5% SLS were used as a positive control,

and deionized water as a negative control

did not induce allergic contact dermatitis

no reactions were observed at induction or

challenge

Abbreviations: human repeated insult patch test (HRIPT); sodium lauryl sulfate (SLS)

Table 14. Case reports with Rosmarinus officinalis (rosemary)

Mode of Contact

Indication

Patch Testing

Reference

cosmetics and cleansing gel con-

taining 0.1% Rosmarinus offici-

nalis (rosemary) leaf extract

itchy erythema of the face; red papules

around the eyes and on the nose and

cheeks

patch test with cosmetics and 1% aq. cleansing gel gave

positive result (+) to gel only on D3

patch tested gel ingredients, only positive reaction (+)

was to 0.1% aq. Rosmarinus officinalis (rosemary) leaf

extract on D3

occupational exposure to a Ros-

marinus officinalis (rosemary)

leaf extract

severe hand, forearm, and face

dermatitis

patch tested with 5 and 10% extract in petrolatum; + re-

action to 5 and 10% on D2 and D5; 1 control was

negative

- patch tested with carnosol in ethanol; ?+ reaction to

0.1% at 3 and D7, + reaction to 1% on D3 and D7;

controls were negative to 0.1 (n=110) and 1% (n=116)

carnosol

occupational use of essential

aromatherapy oils (5 cases)

hand eczema in all; other involvement

seen

- patch testing with the European baseline series, fra-

grance series, and 2% of each essential oil in petro-

latum; ++ reaction to rosemary oil in 2 subjects, + in

one, among other positive reactions

history of eating foods spiced

with rosemary

severe cheilitis

patch tested with 41 antigens, 21 flavoring agents and

dyes, and medications; ++ on D2 and + on D5 to rose-

mary (also + to nickel on D2 and D5; + to wood tars on

D2)

picked rosemary leaves

developed hand, forearm, and face

dermatitis within hours

prick-by-prick testing was negative at 15 min and

positive (++) at D2

- patch testing gave positive reactions with rosemary

(++) and thyme (+) on D2 and D4

- a photopatch test (10 J/cm) with rosemary and thyme

showed stronger reactions (+++ and ++, respectively,

on D4)

- 5 controls were negative

walked near, and touched,

odorous plants

cutaneous lesions on the hand and face;

developed edema and eczematous

lesions on her hands, eyelids, and face

patch and photopatch test with 1% rosemary extract

was positive (+++)

- patch and photopatch test with rosemary leaves was

positive; more intense with photopatch (++/+++)

- hydrophilic and lipophilic rosemary extracts 10%,

patch and photopatch tests were positive

- patch test with 0.1% carnosol in alcohol was positive

- patch test with sage and oregano were negative

-5 controls were negative with all

rosemary leaf plasters applied to

knee

after 3 days, acute dermatitis in the

application area

positive (++ on D2; +++ on D4) reactions in a patch

test with rosemary leaves, but not thyme, origanum, or

mint

- 10 controls did not react to rosemary leaves

applied a poultice containing

rosemary and thyme

after 24 h, acute, cutaneous, eczematous

lesion on right thigh, with vesicles and

blisters

positive patch test results with the poultice (++ on D2

and D4); rosemary (++ on D2 and D4); thyme (- on D2,

++ on D4); and colophony (+ on D2 and D4); negative

results with arnica, chamomile, and horsetails

- 12 controls were negative with rosemary and thyme

rosemary alcohol applied to chest

swelling of face, chest, and dorsal

aspect of arms, followed by peeling

positive reactions were found in patch test with fresh

Rosmarinus officinalis (rosemary) leaves (+++ on D2,

D3, D4), dry rosemary leaves (+ reaction on D2, D3,

D3), dry leaves wetted with water (+ reaction on D2,

D3, D3), the flower (++ reaction on D2, D3, D3), and

rosemary alcohol ((+ reaction on D2, D3, D3)

- negative reactions to 50% aq. rosemary alcohol

- positive reactions were also found with sage and

lavender

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