4-allylveratrole

Endpoint summary

Description of key information

Key study (oral route, rats): Test method similar to OECD 408 and GLP. The NOEL for methyl eugenol after an oral exposure of 14 weeks was estimated to be 10 mg/kg bw/day in rats.

Key study (oral route, mice): Test method similar to OECD 408 and GLP. The NOEL for methyl eugenol after an oral exposure of 14 weeks was estimated to be 10 mg/kg bw/day in mice.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 1988 - 30 September 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Remarks:

(Food and Drug Administration (FDA) Good Laboratory Practice)

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

(F344/N)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 6 weeks
- Weight at study initiation: 112-119 g (males) and 94-98 g (females)
- Housing: 5 per cage; Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed twice weekly; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed twice weekly; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly.
- Water (e.g. ad libitum): Tap water (Birmingham municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day room fluorescent light

Route of administration:

oral: gavage

Details on route of administration:

Although the principal route of human exposure is via food, the gavage route of administration was used because results of preliminary NTP studies showed methyleugenol in feed to be unpalatable to rats and mice (Battelle, 1997).

Vehicle:

methylcellulose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The vehicle was prepared by mixing methylcellulose and heated, deionized water with a magnetic stirrer to form a 0.5% solution, which was then cooled. Methyleugenol was slowly added to the 0.5% methylcellulose and mixed for 2 minutes using a homogenizer fitted with an anaerobic generator. The anaerobic generator was removed and the mixture was stirred with a magnetic stirrer for 1 hour.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 2, 6, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 ml/kg bw /day
- Lot/batch no. (if required): 874544
- Purity: USP/FCC grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of methyleugenol were conducted at the study laboratory using HPLC at the beginning, midpoint, and end of the 14-week study.
All dose formulations used in the 14-week study were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 days per week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(water and vehicle controls)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 (at 300 mg/kg only 9 males)
10 (special study rats used for clinical pathology (received the same doses for 22 or 23 days))

Control animals:

yes

yes, concurrent vehicle

yes, historical

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded weekly and at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 5 and at week 4 of the special study rats (10 male and 10 female) and from all core study rats surviving to the end of the study.
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: No data
- How many animals: 10 male and 10 female per group and all core study rats surviving to the end of the study.
- Parameters checked and examined: hematocrit; hemoglobin concentration; erythrocyte and reticulocyte counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; platelet count and morphology; leukocyte count and differentials.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 5 and at week 4 of the special study rats and from all core study rats surviving to the end of the study.
- Animals fasted: No data
- How many animals: 10 male and 10 female per group and all core study rats surviving to the end of the study.
- Parameters checked and examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Sperm Motility and Vaginal Cytology: At the end of the study, sperm samples were collected from vehicle control, 30, 100, and 300 mg/kg male rats for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda epididymis, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the study from vehicle control, 30, 100, and 300 mg/kg female rats for vaginal cytology evaluations. The following parameters were evaluated: the estrous cycle lengths and relative frequency of estrous stages.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.

HISTOPATHOLOGY: Yes. Complete histopathology was performed on all core study water control, vehicle control, and 1,000 mg/kg rats.
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the kidney, liver, salivary gland, and stomach of male and female rats and the adrenal gland and spleen of male rats in the 10, 30, 100, and 300 mg/kg groups and the testis and uterus of rats in the 100 and 300 mg/kg groups were examined.

Statistics:

Survival Analyses: Kaplan and Meier (1958) determination; Life table trend test (Tarone 1975); Life table pairwise test (Cox 1972); All reported P values for the survival analyses are two sided.

Calculation of Incidence: For calculation of statistical significance, the incidences of most neoplasms and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence, the denominators consist of the number of animals on which a necropsy was performed. The survival-adjusted rate (based on the Poly-3 method) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences: Fisher exact test (interim evaluations) or Poly-3 test; Poly-3 test (incidences) or Mann-Whitney U test (severities); Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.

Analysis of Continuous Variables: Organ and body weight data analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, plasma concentration, spermatid, and epididymal spermatozoal data analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964); Outlier test Dixon and Massey (1951); Jonckheere´s test to assess significance of dose related trends and to determine if a William´s or Shirley´s test was more appropriate than a Dunnett´s or Dunn´s test; Average severity values with Mann-Whitney U test (Hollander and Wolfe, 1973); Treatment effects by applying a multivariate analysis of variance (Morrison, 1976).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings possibly related to chemical administration included emaciation and urine staining in 100, 300, and 1,000 mg/kg female rats.

Mortality:

no mortality observed

Description (incidence):

All rats survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights and body weight gains of 300 and 1,000 mg/kg males and all dosed groups of females were significantly less than those of the vehicle controls; final mean body weights of 1,000 mg/kg males and females were 30% and 16% less than the vehicle controls, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At week 14, there was a minimal treatment-related decrease in hemoglobin concentrations and hematocrit values in the 300 mg/kg females and 1,000 mg/kg males and females. The hematocrit and hemoglobin decreases would be consistent with an anemia, but there was no corresponding decrease in erythrocyte counts. There was, however, an erythrocyte microcytosis demonstrated by decreased mean cell volumes in the 300 mg/kg males and 1,000 mg/kg males and females; this change also occurred on day 5 and/or week 4. Animals in the 300 and 1,000 mg/kg groups also had decreased mean cell hemoglobin values; this would be consistent with the decreases in mean cell volumes. This suggests that, while there were equal numbers of circulating erythrocytes for animals in the 300 and 1,000 mg/kg groups at week 14, the erythrocytes were smaller, resulting in lower hematocrit and hemoglobin values. There was evidence of a thrombocytosis at all time points, demonstrated by increased platelet counts in the 100 mg/kg or greater groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The serum activities of alanine aminotransferase and sorbitol dehydrogenase were increased in the 100 mg/kg or greater male and female groups at various time points and would be consistent with hepatocellular injury or leakage. Additionally, bile acid concentrations were increased in the 300 and 1,000 mg/kg males at all time points and 300 and 1,000 mg/kg females at weeks 4 and 14 and would be consistent with cholestasis or altered hepatic function. However, serum alkaline phosphatase activity, another marker of cholestasis, was either unaffected or decreased in the same animals. A hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations, occurred in rats in the 300 and 1,000 mg/kg groups at all time points. There were minimal increases of creatinine concentrations in the 300 and 1,000 mg/kg female rats at all time points. However, urea nitrogen, another marker of renal function, was either unaffected or decreased in the same animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights of 100, 300, and 1,000 mg/kg males and 300 and 1,000 mg/kg females and testis weights of 1,000 mg/kg males were significantly greater than those of the vehicle control groups. Thymus weights in all dosed males and in 1,000 mg/kg females were significantly less than those of the vehicle control groups. The decreases in thymus weights and differences in other organ weights were likely secondary to body weight differences.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Chemical-related lesions were observed grossly in the liver of male and female rats, the testis of male rats, and the uterus of female rats administered 1,000 mg/kg. Liver foci were observed in 8 of 10 males and in 1 of 10 females. The testes were enlarged in all dosed male rats; female rats had small (3/10) or thin (3/10) uteri.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were observed between the vehicle and water control groups. Compared to the vehicle control groups, there were significant chemical-related increases in the incidences of microscopic lesions in the liver, glandular stomach, adrenal cortex, submandibular salivary gland, testis, and uterus in dosed rats. Incidences of hepatic lesions were significantly increased in rats administered 300 or 1,000 mg/kg, and the lesions were generally more severe in males than in females. Hepatic lesions with increased incidences included cytologic alteration, cytomegaly, Kupffer cell pigmentation, bile duct hyperplasia, and foci of cellular alteration. Cytologic alteration was the term used to describe individual hepatocytes with altered tinctorial staining qualities (eosinophilic or basophilic) and increased mitotic activity and, in general, was of minimal to moderate severity. Cytomegaly was characterized by minimal to moderate enlargement of periportal hepatocytes. Kupffer cell pigmentation was a minimal change characterized by the accumulation of yellow-gold to green pigment within the cytoplasm of periportal hepatocytes. Bile duct hyperplasia consisted of minimal to moderate proliferation of small bile ductules within portal areas. Foci of cellular alteration consisted of discrete foci of hepatocytes with altered cytoplasmic staining (eosinophilic, basophilic, or mixed) and were morphologically similar to those that occur spontaneously or with chemical exposure.
The incidences of atrophy and chronic inflammation of the mucosa of the glandular stomach were significantly increased in rats administered 300 or 1,000 mg/kg. Lesions were generally of minimal to mild severity in the 300 mg/kg groups and mild to moderate in the 1,000 mg/kg groups. Atrophy consisted of a decrease in the thickness of the gastric mucosa due to generalized loss of glandular epithelial parietal and chief cells accompanied by condensation of the lamina propria. Inflammation was of mild severity and consisted of fibrosis and a diffuse infiltration of the lamina propria by lymphocytes, neutrophils, and macrophages. In addition, there was mild glandular dilatation and increased mitotic activity in the glandular epithelial cells.
The incidences of cortical hypertrophy of the adrenal cortex were significantly increased in 100, 300, and 1,000 mg/kg males and 1,000 mg/kg females. The incidences of cytoplasmic alteration of the submandibular salivary glands were increased in rats administered 30 mg/kg or greater.
Cytoplasmic alteration of the submandibular salivary gland consisted of a loss of cytoplasmic zymogen granules with reduction in the size of serous cells and their ducts. Male rats administered 1,000 mg/kg had significantly increased incidences of moderate dilatation of the seminiferous tubules and testicular degeneration characterized by diffuse loss of spermatogenic cells within the seminiferous tubules. Spermatogonia remaining within the seminiferous and epididymal tubules were morphologically normal. The incidences of mild uterine atrophy were significantly increased in female rats administered 300 or 1,000 mg/kg.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One hepatocellular adenoma was present in a male rat administered 1,000 mg/kg.

Other effects:

no effects observed

Description (incidence and severity):

SPERM MOTILITY AND VAGINAL CYTOLOGY: No significant differences in sperm motility or in vaginal cytology parameters between dosed and vehicle control groups were observed.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Survival and Body Weights of Rats in the 14-Week Gavage Study of Methyleugenol

		Mean Body Weight^b(g)
Dose (mg/kg)	Survival^a	Initial	Final	Change	Final Weight Relative to Vehicle Control (%)
Male
Water Control	10/10	114 ± 4	327 ± 6	214 ± 4	99
Vehicle Control	10/10	118 ± 4	331 ± 7	214 ± 5
10	10/10	114 ± 4	331 ± 5	218 ± 5	100
30	10/10	117 ± 5	332 ± 8	216 ± 4	100
100	10/10	119 ± 4	322 ± 7	203 ± 4	97
300	9/9	116 ± 5	304 ± 4**	188 ± 4**	92
1000	10/10	112 ± 4	231 ± 4**	119 ± 5**	70
Female
Water Control	10/10	96 ± 2	192 ± 3	96 ± 3	98
Vehicle Control	10/10	97 ± 2	196 ± 4	99 ± 5
10	10/10	94 ± 2	184 ± 1*	89 ± 2*	93
30	10/10	98 ± 3	187 ± 4*	89 ± 3*	95
100	10/10	97 ± 2	183 ± 2**	86 ± 2**	93
300	10/10	97 ± 3	180 ± 2**	83 ± 3**	92
1000	10/10	95 ± 3	164 ± 4**	69 ± 3**	84

* Significantly different (P≤0.05) from the vehicle control group by Williams’ or Dunnett’s test

** P≤0.01

a Number of animals surviving at 14 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error.

Table 2. Incidences of Selected Neoplasms and Nonneoplastic Lesions in Male and Female Rats in the 14-Week Gavage Study of Methyleugenol

	Water Control	Vehicle Control	10 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Male
Liver^a	10	10	10	10	10	9	10
Cytologic Alteration^b	0	0	0	0	0	9** (1.2)^c	10** (3.0)
Cytomegaly	0	0	0	0	0	9** (1.7)	10** (2.8)
Pigment, Kupffer Cell	0	0	0	0	0	0	10** (1.0)
Basophilic Focus	0	0	0	0	0	0	3 (2.3)
Mixed Cell Focus	0	0	0	0	0	2   (1.5)	9** (2.2)
Hyperplasia, Bile Duct	0	0	0	0	0	6** (1.0)	10** (2.1)
Hepatocellular Adenoma	0	0	0	0	0	0	1
Glandular Stomach	10	10	10	10	10	9	10
Atrophy	0	0	0	0	0	7** (1.7)	10** (3.0)
Inflammation, Chronic	0	0	0	0	0	9** (1.2)	10** (2.6)
Adrenal Cortex	10	10	10	10	10	9	10
Hypertrophy	0	0	0	0	4* (1.0)	9** (1.0)	10** (1.0)
Submandibular Salivary Gland	10	10	10	10	10	9	10
Cytoplasmic Alteration	0	0	0	3  (1.0)	10**  (1.0)	9** (1.0)	10** (1.0)
Testis	10	10	10	10	10	9	10
Dilatation	0	0	0	0	0	0	10** (2.7)
Degeneration	0	0	0	0	0	0	10** (2.8)
Female
Liver	10	10	10	10	10	10	10
Cytologic Alteration	0	0	0	0	0	0	10** (2.3)
Cytomegaly	0	0	0	0	0	9** (1.1)	10** (2.2)
Pigment, Kupffer Cell	0	0	0	0	0	0	9**  (1.0)
Mixed Cell Focus	0	0	0	0	0	2    (1.0)	8**  (1.9)
Hyperplasia, Bile Duct	0	0	0	0	0	0	9**  (1.2)
Glandular Stomach	10	10	10	10	10	10	10
Atrophy	0	0	0	0	0	9**  (1.0)	10** (2.9)
Inflammation, Chronic	0	0	0	0	6** (1.0)	10** (1.9)	10** (2.3)
Adrenal Cortex	10	10	10	10	10	10	10
Hypertrophy	0	2 (1.0)	0	0	0	0	9** (1.0)
Submandibular Salivary Gland	10	10	10	10	10	10	10
Cytoplasmic Alteration	0	0	0	7** (1.0)	10** (1.0)	10** (1.0)	10** (1.0)
Uterus	10	10	10	10	10	10	10
Atrophy	0	0	0	0	0	4*  (2.5)	10** (2.0)

* Significantly different (P≤0.05) from the vehicle control group by the Fisher exact test

** P≤0.01

a Number of animals with tissue examined microscopically

b Number of animals with lesion

c Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Conclusions:

The NOEL of methyl eugenol after an oral exposure of 14 weeks was estimated at 10 mg/kg bw/day in rats.

Executive summary:

A 14-week repeated dose toxicity study (oral route) was conducted to characterize the toxic responses in rats orally exposed to methyl eugenol following a method comparable to OECD TG 408 and in compliance with GLP. Groups of 10 male and 10 female F344/N rats were administered 0, 10, 30, 100, 300, or 1,000 mg/kg body weight in 0.5% methylcellulose by gavage 5 days per week for 14 weeks. Additional groups of rats of each sex were dosed similarly and used for hematology and clinical chemistry studies. A water control group of 10 male and 10 female rats received deionized water by gavage. All rats survived until the end of the study. Methyleugenol administration to rats induced erythrocyte microcytosis in 300 mg/kg males and 1,000 mg/kg males and females, and thrombocytosis in the 100 mg/kg or greater groups. It also caused an increase in serum alanine aminotransferase and sorbitol dehydrogenase activities in the 100 mg/kg or greater rats, and bile acid concentration in the 300 and 1,000 mg/kg groups, suggesting hepatocellular injury, cholestasis or altered hepatic function. Additionally, methyleugenol induced hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations in the 300 and 1,000 mg/kg groups. Liver weights of 100, 300, and 1,000 mg/kg males and 300 and 1,000 mg/kg females and testis weights of 1,000 mg/kg males were significantly increased. Increased incidences of liver lesions occurred in 300 and 1,000 mg/kg males and females and hepatocellular adenoma occurred in one 1,000 mg/kg male. The incidences of atrophy and chronic inflammation of the mucosa of the glandular stomach were significantly increased in rats administered 300 or 1,000 mg/kg. The incidences of cortical hypertrophy of the adrenal cortex were significantly increased in 100, 300, and 1,000 mg/kg males and 1,000 mg/kg females. The incidences of cytoplasmic alteration of the submandibular salivary glands were increased in rats administered 30 mg/kg or greater. Based on the results of this study, the NOEL was estimated to be 10 mg/kg bw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 1988 - 13 October 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Remarks:

(Food and Drug Administration (FDA) Good Laboratory Practice)

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Inc. (Gilroy, CA)
- Age at study initiation: 6 weeks (female), 7 weeks (male)
- Weight at study initiation: 21.1-23.2 g (males) and 18.4-19.5 g (females)
- Housing: 1 per cage; Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly.
- Water (e.g. ad libitum): Tap water (Birmingham municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum.
- Acclimation period: 11 days (female) or 10 days (male)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): minimum of 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day room fluorescent light

Route of administration:

oral: gavage

Details on route of administration:

Although the principal route of human exposure is via food, the gavage route of administration was used because results of preliminary NTP studies showed methyleugenol in feed to be unpalatable to rats and mice (Battelle, 1997).

Vehicle:

methylcellulose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The vehicle was prepared by mixing methylcellulose and heated, deionized water with a magnetic stirrer to form a 0.5% solution, which was then cooled. Methyleugenol was slowly added to the 0.5% methylcellulose and mixed for 2 minutes using a homogenizer fitted with an anaerobic generator. The anaerobic generator was removed and the mixture was stirred with a magnetic stirrer for 1 hour.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 1, 3, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 ml/kg bw /day
- Lot/batch no. (if required): 874544
- Purity: USP/FCC grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of methyleugenol were conducted at the study laboratory using HPLC at the beginning, midpoint, and end of the 14-week study.
All dose formulations used in the 14-week study were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 days per week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

(water and vehicle controls)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes

yes, concurrent vehicle

yes, historical

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded weekly and at the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Sperm Motility and Vaginal Cytology: At the end of the study, sperm samples were collected from vehicle control, 10, 30, and 100 mg/kg male mice for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda epididymis, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the study from vehicle control, 10, 30, and 100 mg/kg female mice for vaginal cytology evaluations. The following parameters were evaluated: the estrous cycle lengths and relative frequency of estrous stages.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.

HISTOPATHOLOGY: Yes. Complete histopathology was performed on all water control, vehicle control, and 300 and 1,000 mg/kg mice.
In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland (with adjacent skin), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the stomach of male and female mice in the 10, 30, and 100 mg/kg groups, the liver of 100 mg/kg male mice and 10, 30, and 100 mg/kg female mice, and the nose of 100 mg/kg male and female mice were examined.

Statistics:

Survival Analyses: Kaplan and Meier (1958) determination; Life table trend test (Tarone 1975); Life table pairwise test (Cox 1972); All reported P values for the survival analyses are two sided.

Calculation of Incidence: For calculation of statistical significance, the incidences of most neoplasms and all nonneoplastic lesions are given as the numbers of animals affected at each site examined microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues before microscopic evaluation, or when neoplasms had multiple potential sites of occurrence, the denominators consist of the number of animals on which a necropsy was performed. The survival-adjusted rate (based on the Poly-3 method) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences: Fisher exact test (interim evaluations) or Poly-3 test; Poly-3 test (incidences) or Mann-Whitney U test (severities); Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.

Analysis of Continuous Variables: Organ and body weight data analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, plasma concentration, spermatid, and epididymal spermatozoal data analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964); Outlier test Dixon and Massey (1951); Jonckheere´s test to assess significance of dose related trends and to determine if a William´s or Shirley´s test was more appropriate than a Dunnett´s or Dunn´s test; Average severity values with Mann-Whitney U test (Hollander and Wolfe, 1973); Treatment effects by applying a multivariate analysis of variance (Morrison, 1976).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical finding was toxicity manifested as generalized morbidity in male and female mice administered 1,000 mg/kg.

Mortality:

mortality observed, treatment-related

Description (incidence):

All mice administered 1,000 mg/kg, except for one male, died before the end of the study. Additionally, one 300 mg/kg male died during week 3 and one 10 mg/kg female died during week 12.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight gains of males and females in the 300 mg/kg groups were significantly less than those of the vehicle controls; the final mean body weights and body weight gains of other groups of mice surviving until the end of the study were similar to those of the vehicle controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The liver weights of 30, 100, and 300 mg/kg males and of 300 mg/kg females were significantly greater than those of the vehicle controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant chemical-related gross lesions were observed in the liver of male and female mice administered 1,000 mg/kg. One male and two females had enlarged livers, one male had a liver nodule, and one female had a pale liver.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Chemical-related nonneoplastic lesions occurred in the liver, glandular stomach, and nose of male and female mice. Hepatic changes were generally only observed in mice that survived beyond the first week of the study. Cytologic alteration, necrosis, bile duct hyperplasia, and focal subacute inflammation occurred in the liver of all 300 mg/kg females. The incidences of these lesions were also significantly increased in 1,000 mg/kg males and females (except subacute inflammation). Female mice administered 1,000 mg/kg had lower incidences of these lesions due to early mortality. Significant differences were not observed between the water and vehicle control mice. Cytologic alteration was observed primarily in periportal sites and was the term used to describe a variety of hepatocellular alterations that included mild nuclear and cytoplasmic enlargement (hypertrophy) and increased cytoplasmic eosinophilia. Necrosis of scattered individual hepatocytes occurred throughout the hepatic lobules. Bile duct hyperplasia consisted of proliferation of immature biliary cells within portal areas. Inflammation consisted of multiple small foci of primarily mononuclear inflammatory cells randomly scattered throughout the liver.

The incidences of atrophy, degeneration, necrosis, edema, mitotic alteration, and cystic glands of the fundic region of the glandular stomach were increased in one or more groups of male and female mice administered 30 mg/kg or greater compared to the vehicle controls; these lesions were generally of minimal to mild severity. In males, the incidences of cystic glands in the 30 mg/kg group and degeneration and mitotic alteration in the 300 mg/kg group were significantly increased. In female mice administered 300 mg/kg, the incidences of atrophy, degeneration, edema, mitotic alteration, and cystic glands were significantly increased. In the 1,000 mg/kg mice, early death and subsequent autolysis significantly precluded adequate histopathologic evaluation of gastric lesions. Lesions were generally of minimal to mild severity in the 30, 100, and 300 mg/kg groups and of mild to marked severity in the 1,000 mg/kg groups. Atrophy consisted of a generalized decrease in the thickness of the mucosal epithelium due to loss of parietal and chief cells and shortening of the mucosal glands. Necrosis in the glandular epithelium was characterized by necrosis of parietal cells primarily, and to a lesser extent, chief cells. Degeneration consisted of dilated glands lined by dysplastic atypical epithelial cells and cellular detritus. Cystic glands were dilated and lined by flattened epithelium. Increased numbers of morphologically normal mitotic figures were present in regenerative areas of the glandular epithelium. Edema was of minimal to mild severity and occurred in the lamina propria.

Minimal to mild focal degeneration of the olfactory epithelium of the nose occurred in 30, 300, and 1,000 mg/kg males, 30 mg/kg or greater females, and two water control females. The incidences and severities of this lesion were not dose dependent. Degeneration consisted of unilateral or bilateral focal loss and/or disruption of the sensory olfactory epithelial cells in the dorsal meatus

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SPERM MOTILITY AND VAGINAL CYTOLOGY: Male mice administered 10 or 30 mg/kg had significantly lower cauda epididymis, epididymis, and testis weights than did the vehicle controls. Additionally, 100 mg/kg males had significantly decreased spermatozoal concentrations. There were no significant differences in vaginal cytology parameters between dosed and vehicle control mice.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Survival and Body Weights of Mice in the 14-Week Gavage Study of Methyleugenol

		Mean Body Weight^b(g)
Dose (mg/kg)	Survival^a	Initial	Final	Change	Final Weight Relative to Vehicle Control (%)
Male
Water Control	10/10	22.7 ± 0.5	32.1 ± 1.2	9.4 ± 1.1	96
Vehicle Control	10/10	22.3 ± 0.4	33.4 ± 0.7	11.1 ± 0.5
10	10/10	22.9 ± 0.4	33.4 ± 0.8	10.5 ± 0.5	100
30	10/10	22.3 ± 0.3	32.6 ± 0.8	10.3 ± 0.6	98
100	10/10	23.2 ± 0.5	33.7 ± 0.9	10.5 ± 0.7	101
300	9/10^c	22.3 ± 0.4	30.7 ± 0.6	8.4 ± 0.5**	92
1000	1/10^d	21.1 ± 0.7	27.3	6.2	82
Female
Water Control	10/10	19.0 ± 0.4	30.9 ± 1.1	11.8 ± 0.9	104
Vehicle Control	10/10	19.0 ± 0.3	29.6 ± 0.6	10.7 ± 0.5
10	9/10^e	18.5 ± 0.3	29.2 ± 0.7	10.7 ± 0.7	99
30	10/10	19.4 ± 0.4	29.3 ± 0.7	9.9 ± 0.6	99
100	10/10	19.5 ± 0.2	29.2 ± 0.5	9.6 ± 0.5	99
300	10/10	19.4 ± 0.2	27.5 ± 0.6	8.1 ± 0.4**	93
1000	0/10^f	18.4 ± 0.3	-	-	-

** Significantly different (P≤0.01) from the vehicle control group by Williams’ or Dunnett’s test

a Number of animals surviving at 14 weeks/number initially in group

b Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study. No standard error was calculated for groups with high mortality. No final mean body weights or weights changes were calculated for groups with 100% mortality.

c Week of death: 3

d Weeks of death: 1, 1, 1, 1, 1, 5, 8, 9, 12

e Week of death: 12

f Weeks of death: 1, 1, 1, 1, 1, 1, 3, 4, 4, 6

Table 2. Incidences of Selected Neoplasms and Nonneoplastic Lesions in Male and Female Mice in the 14-Week Gavage Study of Methyleugenol

	Water Control	Vehicle Control	10 mg/kg	30 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Male
Liver^a	10	10	10	10	10	10	10
Cytologic Alteration^b	0	0	0	0	0	0	5** (4.0)^c
Necrosis	0	0	0	0	0	0	5** (2.0)
Hyperplasia, Bile Duct	0	0	0	0	0	0	5** (2.2)
Inflammation, Subacute	0	0	0	0	0	0	4** (1.8)
Glandular Stomach	10	10	10	10	10	10	10
Atrophy	0	0	0	0	0	1 (1.0)	3 (4.0)
Degeneration	0	0	0	1 (1.0)	1 (1.0)	9** (1.0)	3 (2.0)
Necrosis	0	0	0	0	0	0	3 (3.3)
Edema	0	0	0	0	0	0	3 (1.3)
Mitotic Alteration	0	0	0	1 (1.0)	0	10** (1.1)	0
Cystic Glands	0	0	0	6** (1.0)	0	2 (1.0)	1 (2.0)
Nose
Epithelial Cell, Degeneration	0	0	0	4** (1.5)	0	1 (1.0)	5** (1.8)
Female
Liver	10	10	10	10	10	10	10
Cytologic Alteration	0	0	0	1 (1.0)	0	10** (2.3)	4** (3.8)
Necrosis	0	0	0	0	0	10** (1.0)	4** (1.8)
Hyperplasia, Bile Duct	0	0	0	0	0	10** (2.0)	4** (1.5)
Inflammation, Subacute	0	0	0	0	0	10** (1.0)	1 (2.0)
Glandular Stomach	10	10	10	10	10	10	10
Atrophy	0	0	0	0	0	10** (1.3)	3 (4.0)
Degeneration	0	0	0	4** (1.0)	3 (1.0)	10** (1.7)	3 (2.0)
Necrosis	0	0	0	0	0	0	3 (4.0)
Edema	0	0	0	0	0	6** (1.0)	3 (1.7)
Mitotic Alteration	0	0	0	4** (1.0)	5** (1.0)	10** (1.2)	0
Cystic Glands	0	0	0	4** (1.5)	0	7** (1.0)	0
Nose
Epithelial Cell, Degeneration	2 (1.0)	0	0	8** (1.0)	4** (1.0)	5** (1.0)	3 (1.3)

* Significantly different (P≤0.05) from the vehicle control group by the Fisher exact test

** P≤0.01

a Number of animals with tissue examined microscopically

b Number of animals with lesion

c Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Conclusions:

The NOEL of methyl eugenol after an oral exposure of 14 weeks was estimated at 10 mg/kg bw/day in mice.

Executive summary:

A 14-week repeated dose toxicity study (oral route) was conducted to characterize the toxic responses in mice orally exposed to methyl eugenol following a method comparable to OECD TG 408 and in compliance with GLP. Groups of 10 male and 10 female B6C3F1 mice were administered 0, 10, 30, 100, 300, or 1,000 mg/kg body weight in 0.5% methylcellulose by gavage 5 days per week for 14 weeks. A water control group of 10 male and 10 female mice received deionized water by gavage. All but one male and all females receiving 1,000 mg/kg died before the end of the study. Chemical-related nonneoplastic lesions occurred in the liver, glandular stomach, and nose of male and female mice. Hepatic changes were generally only observed in mice that survived beyond the first week of the study. Cytologic alteration, necrosis, bile duct hyperplasia, and focal subacute inflammation occurred in the liver of all 300 mg/kg females. The incidences of atrophy, degeneration, necrosis, edema, mitotic alteration, and cystic glands of the fundic region of the glandular stomach were increased in one or more groups of male and female mice administered 30 mg/kg or greater compared to the vehicle controls. Minimal to mild focal degeneration of the olfactory epithelium of the nose occurred in 30, 300, and 1,000 mg/kg males, 30 mg/kg or greater females, and two water control females, although incidences and severities of this lesion were not dose dependent. Based on the results of this study, the NOEL was estimated to be 10 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

2 studies with Klimisch score 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study (oral route, rats): A 14-week repeated dose toxicity study (oral route) was conducted to characterize the toxic responses in rats orally exposed to methyl eugenol following a method comparable to OECD TG 408 and in compliance with GLP. Groups of 10 male and 10 female F344/N rats were administered 0, 10, 30, 100, 300, or 1,000 mg/kg body weight in 0.5% methylcellulose by gavage 5 days per week for 14 weeks. Additional groups of rats of each sex were dosed similarly and used for hematology and clinical chemistry studies. A water control group of 10 male and 10 female rats received deionized water by gavage. All rats survived until the end of the study. Methyleugenol administration to rats induced erythrocyte microcytosis in 300 mg/kg males and 1,000 mg/kg males and females, and thrombocytosis in the 100 mg/kg or greater groups. It also caused an increase in serum alanine aminotransferase and sorbitol dehydrogenase activities in the 100 mg/kg or greater rats, and bile acid concentration in the 300 and 1,000 mg/kg groups, suggesting hepatocellular injury, cholestasis or altered hepatic function. Additionally, methyleugenol induced hypoproteinemia and hypoalbuminemia, evidenced by decreased total protein and albumin concentrations in the 300 and 1,000 mg/kg groups. Liver weights of 100, 300, and 1,000 mg/kg males and 300 and 1,000 mg/kg females and testis weights of 1,000 mg/kg males were significantly increased. Increased incidences of liver lesions occurred in 300 and 1,000 mg/kg males and females and hepatocellular adenoma occurred in one 1,000 mg/kg male. The incidences of atrophy and chronic inflammation of the mucosa of the glandular stomach were significantly increased in rats administered 300 or 1,000 mg/kg. The incidences of cortical hypertrophy of the adrenal cortex were significantly increased in 100, 300, and 1,000 mg/kg males and 1,000 mg/kg females. The incidences of cytoplasmic alteration of the submandibular salivary glands were increased in rats administered 30 mg/kg or greater. Based on the results of this study, the NOEL was estimated to be 10 mg/kg bw/day.

Key study (oral route, mice): A 14-week repeated dose toxicity study (oral route) was conducted to characterize the toxic responses in mice orally exposed to methyl eugenol following a method comparable to OECD TG 408 and in compliance with GLP. Groups of 10 male and 10 female B6C3F1 mice were administered 0, 10, 30, 100, 300, or 1,000 mg/kg body weight in 0.5% methylcellulose by gavage 5 days per week for 14 weeks. A water control group of 10 male and 10 female mice received deionized water by gavage. All but one male and all females receiving 1,000 mg/kg died before the end of the study. Chemical-related nonneoplastic lesions occurred in the liver, glandular stomach, and nose of male and female mice. Hepatic changes were generally only observed in mice that survived beyond the first week of the study. Cytologic alteration, necrosis, bile duct hyperplasia, and focal subacute inflammation occurred in the liver of all 300 mg/kg females. The incidences of atrophy, degeneration, necrosis, edema, mitotic alteration, and cystic glands of the fundic region of the glandular stomach were increased in one or more groups of male and female mice administered 30 mg/kg or greater compared to the vehicle controls. Minimal to mild focal degeneration of the olfactory epithelium of the nose occurred in 30, 300, and 1,000 mg/kg males, 30 mg/kg or greater females, and two water control females, although incidences and severities of this lesion were not dose dependent. Based on the results of this study, the NOEL was estimated to be 10 mg/kg bw/day.

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.