4-allylveratrole

Carcinogenicity

Description of key information

Key study. Test method comparable to OECD TG 451, GLP study. There was clear evidence of carcinogenic activity of methyleugenol in male and female F344/N rats.

Key study. Test method comparable to OECD TG 451, GLP study. There was clear evidence of carcinogenic activity of methyleugenol in male and female B6C3F1 mice.

Supporting study. Hepatocarcinogenicity study in B6C3F1 male mice. There was clear evidence of the development of hepatomas in mice after i.p. injections prior to weaning.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 1994 to February 1996

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 451 (Carcinogenicity Studies)

GLP compliance:

yes

Remarks:

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

Species:

rat

Strain:

Fischer 344

Remarks:

(F344/N)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: 124-127 g (males) and 106-108 g (females) recorded on first week of the study.
- Fasting period before study: not specified
- Housing: 3 (males) or 5 (females) 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): ad libitum. NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly.
- Water (e.g. ad libitum): ad libitum. Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI).
- Acclimation period: 11 days (males) or 12 days (females).

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on 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 0.5% methylcellulose and then diluted to the required volume with additional 0.5% methylcellulose while being stirred continuously.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 7.4, 15, 30 or 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): 876672 and 946150
- Purity: not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed with ultraviolet spectroscopy approximately every 8 weeks. All dose formulations used in the study were within 11% of the target concentrations.

Duration of treatment / exposure:

105 weeks, except 300 mg/kg group, which received only the 0.5% methylcellulose vehicle from week 53 to study completion

Frequency of treatment:

5 days per week

Post exposure period:

No

Dose / conc.:

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

Remarks:

(vehicle control)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

50 (37, 75, and 150 mg/kg)
60 (vehicle control and 300 mg/kg)

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: Based on lower mean body weights and increased incidences of nonneoplastic liver and glandular stomach lesions in 300 and 1,000 mg/kg rats in a 14-week study, methyleugenol doses selected for rats in the 2-year gavage study were 37, 75, and 150 mg/kg. Stop-exposure groups were given 300 mg/kg for 12 months to provide additional data on progression and regression of liver lesions.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded every 4 weeks and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at the beginning of the studies, every 4 weeks, and at the end of the studies.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, performed on all animals.

HISTOPATHOLOGY: Yes, performed on all animals.
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.

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:

no effects observed

Description (incidence and severity):

There were no clinical findings that could be attributed to methyleugenol administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All 150 and 300 mg/kg males died before the end of the study, and survival of 150 mg/kg females was slightly less than that of the vehicle controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males and females were less than compared to vehicle control throughout most of the study. There was some recovery in mean body weights in the 300 mg/kg groups when dosing was discontinued for 12 months.

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:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions were observed in the liver and stomach of males and females. Focal areas of discoloration, nodules (raised lesions less than 5 mm in diameter), and masses (raised lesions greater than 5 mm in diameter) were observed in the livers of dosed rats. The incidences and multiplicity of focal areas of discoloration, nodules, and masses increased with increasing dose. Grossly, there was diffuse thickening of the entire glandular portion of the stomach with or without the presence of one or more nodules and masses.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Chemical-related lesions included eosinophilic, basophilic, and mixed cell foci; oval cell and bile duct hyperplasia; cystic degeneration; and hepatocyte hypertrophy in core study and 300 mg/kg (stop-exposure) rats.
Glandular Stomach: Chemical-related nonneoplastic lesions occurred in the glandular stomach of male and female rats. Lesions were confined to the fundic region and were more prevalent and severe in females than males. Lesions included glandular epithelial atrophy and neuroendocrine cell hyperplasia.
Kidney: The incidences of nephropathy were increased in all dosed groups of females, and the increase was significant in the 300 mg/kg group. The incidences of nephropathy in dosed groups of males were similar to that in the vehicle controls. In dosed males, the severity of nephropathy increased with increasing dose; the severity in 300 mg/kg males was significantly greater than that in the vehicle controls.
Bone Marrow: Bone marrow hyperplasia was observed at 6 months in one male and four females administered 300 mg/kg and at 12 months in one vehicle control male and five 300 mg/kg males. At 2 years, the incidences of bone marrow hyperplasia were increased in all groups of dosed females (4/50, 15/50, 11/49, 20/50, 25/50), and the increases in the 37, 150, and 300 mg/kg groups were significant. Bone marrow hyperplasia consisted of a marked increase in the density of erythroid or myeloid cells or a mixture of both, frequently accompanied by proliferation of megakaryocytes. Bone marrow hyperplasia was considered a reactive response secondary to significant hepatic and gastric pathology.
Salivary Gland: The incidences of cytoplasmic alteration of the submandibular salivary gland of all dosed male and female rats were significantly greater than those in the vehicle controls (males: vehicle control, 4/50; 37 mg/kg, 50/50; 75 mg/kg, 49/50; 150 mg/kg, 48/48; 300 mg/kg, 48/48; females: 1/50, 48/48, 49/49, 49/49, 49/50). This change was also present in all 300 mg/kg rats at 6 and 12 months. Cytoplasmic alteration consisted of a loss of the eosinophilic granules within the striated ducts of the submandibular salivary glands.
Adrenal medulla: Incidences of hyperplasia did not differ between vehicle control and dosed groups of either male or female rats.
Spleen: The incidences of splenic fibrosis in 150 and 300 mg/kg females at 2 years were significantly increased (3/50, 3/50, 5/50, 12/49, 15/50).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Chemical-related lesions included hepatocellular adenoma; hepatocellular carcinoma; hepatocholangioma and hepatocholangiocarcinoma in core study and 300 mg/kg (stop-exposure) rats.
Glandular Stomach: Chemical-related neoplasms lesions occurred in the glandular stomach of male and female rats. Lesions were confined to the fundic region and were more prevalent and severe in females than males. Lesions included benign and malignant neuroendocrine tumors.
Forestomach: In core study female rats, there was a positive trend in the incidences of squamous cell papilloma or carcinoma (combined) (0/50, 0/50, 1/50, 3/50); however, by pairwise comparisons, the incidences in the dosed groups were not significantly greater than that in the vehicle control group. The incidence in the 150 mg/kg group exceeded the historical control range for corn oil gavage vehicle controls.
Kidney: In the standard evaluation in core study male rats, there was a positive trend in the incidences of renal tubule adenoma, and the incidence in the 300 mg/kg males was significantly greater than that in the vehicle controls. The incidences in all groups exceeded the historical control range for corn oil gavage vehicle controls. The incidences of renal tubule proliferative lesions in male rats were suggestive of a neoplastic effect in the kidney. Therefore, additional step sections of the kidneys of male rats were prepared using the residual formalin-fixed wet tissue. During this evaluation, additional rats with renal tubule proliferative lesions were identified. The incidences of renal tubule hyperplasia and adenoma in the extended evaluation and the combined incidences of standard and step sections in the 75, 150, and 300 mg/kg groups were greater than those in the vehicle controls. Additionally, there was a slight positive trend in the incidences of renal tubule benign oncocytoma in the extended evaluation and the combined incidences of standard and step sections.
Malignant Mesothelioma: In core study males, there was a positive trend in the incidences of malignant mesothelioma (vehicle control, 1/50; 37 mg/kg, 3/50; 75 mg/kg, 5/50; 150 mg/kg, 12/50); the incidence was significantly greater in 150 mg/kg males than in the vehicle controls. The incidence in 300 mg/kg males (5/50) was also significantly greater than that in the vehicle controls. The incidences in the 75, 150, and 300 mg/kg groups exceeded the historical control range for corn oil gavage studies. Mesotheliomas were disseminated along the peritoneal surface of several organs in the abdominal cavity and/or the serosa of the testis and epididymis.
Mammary Gland: Mammary gland fibroadenoma occurred with a positive trend in male rats. The incidences of mammary gland fibroadenoma in 75 and 150 mg/kg males were significantly greater than in the vehicle controls (5/50, 5/50, 15/50, 13/50, 6/50). The incidences of mammary gland fibroadenoma in all groups of males exceeded the historical control range for corn oil gavage vehicle controls [23/402 (5.7% ± 1.3%); range, 4%-8%].
Skin (subcutaneous tissue): The incidences of fibroma in 37 and 75 mg/kg males (1/50, 9/50, 8/50, 5/50, 4/50) and combined incidences of fibroma or fibrosarcoma in 37, 75, and 150 mg/kg males (1/50, 12/50, 8/50, 8/50, 4/50) were significantly increased; however, the incidences did not increase with increasing dose. The incidences of these lesions in these groups exceeded the historical range for corn oil gavage vehicle controls.
Adrenal Medulla: At 2 years, there was a negative trend in the incidences of benign pheochromocytoma in core study males, and the incidences were significantly decreased in the 75 and 150 mg/kg groups compared to the vehicle controls (24/50, 17/50, 11/50, 10/50, 9/50). However, the incidence in the concurrent control group (48%) is high compared to the historical control rate for corn oil gavage studies (23%); the incidence in the concurrent controls is also high compared to the historical control rates for dosed feed (26%) and inhalation (32%) studies (historical control data). The incidences of benign pheochromocytoma in the 75, 150, and 300 mg/kg groups of male rats are also within the historical control range for these routes of administration. Furthermore, there was a significant increase in the incidence of benign pheochromocytoma in 300 mg/kg females at 2 years (1/50, 1/50, 2/50, 2/49, 6/50). Although 6/50 is just outside of the historical control range for female rats in corn oil gavage and dosed feed studies, as many as 6/50 have been seen in chamber control groups from inhalation studies. The decreased incidences of benign pheochromocytoma in male rats and the increased incidence in female rats were considered variations due to chance rather than to effects associated with methyleugenol administration.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

Under the conditions of this study, there was clear evidence of carcinogenic activity of methyleugenol in male and female F344/N rats based on the increased incidences of liver neoplasms and neuroendocrine tumors of the glandular stomach in male and female rats and the increased incidences of kidney neoplasms, malignant mesothelioma, mammary gland fibroadenoma, and subcutaneous fibroma and fibroma or fibrosarcoma (combined) in male rats. A marginal increase in the incidence of squamous cell neoplasms of the forestomach may have been related to methyleugenol administration in female rats.

Key result

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Summary of the 2-Year Carcinogenesis study of Methyleugenol

	Male F344/N Rats	Female F344/N Rats
Doses administered in methylcellulose by gavage	0, 37, 75, or 150 mg/kg or 300 mg/kg (stop-exposure)	0, 37, 75, or 150 mg/kg or 300 mg/kg (stop­exposure)
Body weights	Dosed groups less than the vehicle control group	Dosed groups less than the vehicle control group
Survival rates	20/50, 16/50, 15/50, 0/50, 0/50	22/50, 25/50, 22/50, 11/50, 16/50
Nonneoplastic effects	Liver: eosinophilic foci (11/50, 28/50, 43/50, 47/50, 39/50); mixed cell foci (1/50, 7/50, 14/50, 8/50, 2/50); hepatocyte hypertrophy (0/50, 13/50, 25/50, 30/50, 26/50); oval cell hyperplasia (14/50, 17/50, 24/50, 34/50, 27/50); cystic degeneration (4/50, 2/50, 25/50, 38/50, 41/50) Glandular stomach: neuroendocrine cell hyperplasia (0/50, 0/50, 1/50, 8/50, 8/50); atrophy (0/50, 14/50, 32/50, 37/50, 29/50)	Liver: eosinophilic foci (10/50, 20/50,27/49, 31/49, 37/50); mixed cell foci (6/50, 4/50, 19/49, 9/49, 7/50); hepatocyte hypertrophy (1/50, 13/50, 16/49, 26/49, 31/50); oval cell hyperplasia (1/50, 15/50, 19/49, 35/49, 34/50); bile duct hyperplasia (11/50, 11/50, 17/49, 22/49, 30/50); cystic degeneration (0/50, 0/50, 1/49, 4/49, 29/50) Glandular stomach: neuroendocrine cell hyperplasia (0/50, 5/50, 11/50, 9/50, 3/50); atrophy (3/50, 41/50, 45/50, 39/50, 33/50)
Neoplastic effects	Liver: hepatocellular adenoma (5/50, 12/50, 23/50, 38/50, 32/50);hepatocellular carcinoma (2/50, 3/50, 14/50, 25/50, 36/50);hepatocellular adenoma or carcinoma (7/50, 14/50, 28/50, 43/50, 45/50);hepatocholangioma (0/50, 0/50, 0/50, 1/50, 6/50);hepatocholangiocarcinoma (0/50, 0/50, 1/50, 1/50, 7/50); hepatocholangiomaorhepatocholangiocarcinoma (0/50, 0/50, 1/50, 2/50, 13/50) Glandular stomach: benign neuroendocrine tumor (0/50, 0/50, 0/50, 3/50, 2/50); malignant neuroendocrine tumor (0/50, 0/50, 0/50, 4/50, 2/50); benign or malignant neuroendocrine tumor (0/50, 0/50, 0/50, 7/50, 4/50) Kidney: renal tubule adenoma (standard and extended evaluations combined - 4/50, 6/50, 17/50, 13/50, 20/50) Malignant mesothelioma:(1/50, 3/50, 5/50, 12/50, 5/50) Mammary gland: fibroadenoma (5/50, 5/50, 15/50, 13/50, 6/50) Skin (subcutaneous): fibroma (1/50, 9/50, 8/50, 5/50, 4/50); fibroma or fibrosarcoma (1/50, 12/50, 8/50, 8/50, 4/50)	Liver: hepatocellular adenoma (1/50, 8/50, 11/49, 33/49, 43/50); hepatocellular carcinoma (0/50, 0/50, 4/49, 8/49, 22/50); hepatocellular adenoma or carcinoma (1/50, 8/50, 14/49, 34/49, 43/50); hepatocholangioma (0/50, 0/50, 0/49, 0/49, 8/50); hepatocholangiocarcinoma (0/50, 0/50, 0/49, 3/49, 9/50); hepatocholangioma or hepatocholangiocarcinoma (0/50, 0/50, 0/49, 3/49, 17/50) Glandular stomach: benign neuroendocrine tumor (0/50, 0/50, 13/50, 9/50, 5/50); malignant neuroendocrine tumor (0/50, 1/50, 12/50, 26/50, 36/50); benign or malignant neuroendocrine tumor (0/50, 1/50, 25/50, 34/50, 41/50)
Uncertain findings		Forestomach: squamous cell papilloma or carcinoma (0/50, 0/50, 1/50, 3/50, 1/50)
Level of evidence of carcinogenic activity	Clear evidence	Clear evidence

Table 2: Survival of Rats in the 2-Year Gavage Study of Methyleugenol

	Vehicle Control	37 mg/kg	75 mg/kg	150 mg/kg	300 mg/kg (Stop-Exposure)
Male
Animals initially in study	60	50	50	50	60
6-Month interim evaluation^a	5	0	0	0	5
12-Month interim evaluation^a	5	0	0	0	5
Accidental deaths^a	0	1	3	1	1
Moribund	15	18	15	20	23
Natural deaths	15	15	17	29	26
Animals surviving to study termination	20	16	15	0	0
Percent probability of survival at end of study^b	40	33	32	0	0
Mean survival (days)^c	678	659	646	615	538
Survival analysis^d	P<0.001	P=0.389	P=0.294	P<0.001	P<0.001
Female
Animals initially in study	60	50	50	50	60
6-Month interim evaluation	5	0	0	0	5
12-Month interim evaluation	5	0	0	0	5
Accidental deaths	0	0	1	1	0
Moribund	17	16	14	26	25
Natural deaths	11	9	13	12	9
Animals surviving to study termination	22	25	22	11	16
Percent probability of survival at end of study	44	50	45	23	32
Mean survival (days)	659	672	675	647	638
Survival analysis	P=0.015	P=0.640N	P=0.807N	P=0.053	P=0.343

a Censored from survival analyses

b Kaplan-Meier determinations

c Mean of all deaths (uncensored, censored, and terminal sacrifice)

d The result of the life table trend test (Tarone, 1975) is in the vehicle control column [the 300 mg/kg (stop-exposure) group was excluded from the trend test], and the results of the life table pairwise comparisons (Cox, 1972) with the vehicle controls are in the dosed group columns. A lower mortality in a dose group is indicated by N.

Table 3: Mean Body Weights of Male Rats in the 2-Year Gavage Study of Methyleugenol

Weeks on Study	Vehicle Control	37 mg/kg		75 mg/kg		150 mg/kg		300 mg/kg
	Av. Wt. (g)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)
Mean for weeks
1-13	255	253	99	253	99	250	98	248	98
14-52	414	406	98	397	96	386	93	370	89
53-101	454	427	94	412	91	388	85	376	82

Table 4: Mean Body Weights of Female Rats in the 2-Year Gavage Study of Methyleugenol

Weeks on Study	Vehicle Control	37 mg/kg		75 mg/kg		150 mg/kg		300 mg/kg
	Av. Wt. (g)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)
Mean for weeks
1-13	165	163	99	162	99	162	98	158	96
14-52	246	235	96	226	92	216	88	207	85
53-101	331	306	93	274	83	255	77	251	76

Conclusions:

Under the conditions of this study, there was clear evidence of carcinogenic activity of methyleugenol in male and female F344/N rats.

Executive summary:

A 2-year carcinogenicity study was conducted to characterize the toxic responses in rats orally exposed to methyleugenol following a method comparable to OECD TG 451 and in compliance with GLP. Groups of 50 male and 50 female F344/N rats received methyleugenol in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg, 5 days per week for 105 weeks; groups of 60 male and 60 female rats received the 0.5% methylcellulose vehicle only. Stop-exposure groups of 60 male and 60 female rats received 300 mg/kg in 0.5% methylcellulose by gavage for 52 weeks followed by just the 0.5% methylcellulose vehicle for the remaining 53 weeks of the study. Cage side observations, clinical findings and body weight evolution were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. All 150 and 300 mg/kg males died before the end of the study, and survival of 150 mg/kg females was slightly less than that of the vehicle controls. Mean body weights of all dosed groups of rats were less than those of the vehicle controls throughout most of the study. Methyleugenol administration caused significant increases in the incidences of nonneoplastic lesions of the liver and glandular stomach of both male and female rats. Increased incidences of liver neoplasms and neuroendocrine tumors of the glandular stomach were observed in both male and female rats. Increased incidences of kidney neoplasms, malignant mesothelioma, mammary gland fibroadenoma, and subcutaneous fibroma and fibroma or fibrosarcoma (combined) were observed in male rats. A marginal increase in the incidence of squamous cell neoplasms of the forestomach may have been related to methyleugenol administration in female rats. Based on these results, methyleugenol is suspected of causing cancer through oral exposure.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1993 to October 1995

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 451 (Carcinogenicity Studies)

GLP compliance:

yes

Remarks:

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

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 24.2-24.4 g (males) and 19.1-19.3 g (females) recorded on first week of the study.
- Fasting period before study: not specified
- Housing: 1 (males) or 5 (females) per cage. Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed twice weekly for females and once weekly for males; Bedding: Sani-Chips® (P.J. Murphy Forest Products Corp., Montville, NJ), changed twice weekly for females and once weekly for males; Racks: Stainless steel (Lab Products Inc., Maywood, NJ), changed and rotated every 2 weeks.
- Diet (e.g. ad libitum): ad libitum. NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed weekly.
- Water (e.g. ad libitum): ad libitum. Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI).
- Acclimation period: 11 days (males) or 12 days (females).

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

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose

Details on 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 0.5% methylcellulose and then diluted to the required volume with additional 0.5% methylcellulose while being stirred continuously.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported.
- Concentration in vehicle: 3.7, 7.5 and 15 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): 876672 and 946150
- Purity: not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed with ultraviolet spectroscopy approximately every 8 weeks. All dose formulations used in the study were within 11% of the target concentrations.

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

5 days per week

Post exposure period:

No

Dose / conc.:

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

Remarks:

(vehicle control)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

yes, historical

Details on study design:

- Dose selection rationale: Based on the high mortality rate of the 1000 mg/kg group, the lower mean body weight gains of the 300 mg/kg groups, and the hepatotoxic effects in the 300 and 1000 mg/kg groups in a 14-week study, the highest dose selected in this 2-year gavage study in mice was 150 mg/kg.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded every 4 weeks and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed at the beginning of the studies, every 4 weeks, and at the end of the studies.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, performed on all animals.

HISTOPATHOLOGY: Yes, performed on all animals.
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) (except male mice), 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.

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:

no effects observed

Description (incidence and severity):

There were no clinical findings that could be attributed to methyleugenol administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival of all dosed groups of male mice was similar to that of the vehicle controls. Survival rates of all dosed groups of female mice were significantly less than that of the vehicle controls. The decreased survival in female mice may have been due to the increased incidences of neoplasms and nonneoplastic lesions; these increases were greater in female mice than in males.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights were generally less than those of the vehicle controls after weeks 81, 41, and 17 in males and after weeks 33, 17, and 5 in females for the 37, 75, and 150 mg/kg groups, 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:

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:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Chemical-related increases in the incidences of hepatic neoplasms and nonneoplastic lesions occurred in males and females. Lesions were frequently multi-focal and coexisted within the liver. Chemical-related nonproliferative lesions included chronic active inflammation in males, and hepatocyte necrosis, hemosiderin pigmentation, and hematopoietic cell proliferation in females.
Based on retrospective analyses, Helicobacter hepaticus was determined to have infected mice in 12 recent NTP 2-year studies. Of the 12 studies, mice (primarily males) from nine studies had H. hepaticus-associated hepatitis. In the methyleugenol study, the incidences of one of the lesions typically associated with H. hepaticus infection, oval cell hyperplasia, increased with increasing dose, which is not typical of the incidences in the other nine studies. In those studies, all groups, including control animals, were relatively equally affected. Also in the methyleugenol study, dose-related increases in the incidences of oval cell hyperplasia occurred in female mice and in the rats. F344/N rats have not been shown to be infected with H. hepaticus, and liver lesions in infected female B6C3F1 mice from NTP studies are minimal to nonexistent; therefore, oval cell hyperplasia was attributed to administration of methyleugenol and was not considered to be related to the presence of H. hepaticus. Using Warthin-Starry silver stains on the livers of 12 male mice (two vehicle controls and ten 150 mg/kg males) from the methyleugenol study, no organisms consistent with H. hepaticus were identified. While mice from this study of methyleugenol were considered to be infected with H. hepaticus based on the PCR-RFLP-based assay, liver disease associated with the infection was not apparent, and the infection was not considered to have compromised the outcome of the toxicology and carcinogenesis studies of methyleugenol.
Glandular Stomach: Nonneoplastic lesions observed in male and female mice included glandular ectasia, mucosal atrophy, chronic active inflammation, epithe¬lial hyperplasia, and neuroendocrine cell hyperplasia. Lesions coexisted and were confined to the fundic region of the glandular stomach, affecting both the more superficial foveolar epithelium of the gastric pits and the deeper epithelium of the gastric glands.
Bone Marrow: In male and female mice, the incidences of bone marrow hyperplasia were significantly greater than those in the vehicle controls (males: vehicle control, 14/49; 37 mg/kg, 26/49; 75 mg/kg, 33/50; 150 mg/kg, 35/50; females: 18/50, 47/50, 46/48, 50/50); the incidences in male mice increased with increasing dose. Bone marrow hyperplasia was considered to be secondary to the changes in hepatic and gastric pathology.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Chemical-related increases in the incidences of hepatic neoplasms and nonneoplastic lesions occurred in males and females. Lesions were frequently multi-focal and coexisted within the liver. Chemical-related proliferative lesions included hepatocellular adenoma and carcinoma, hepatoblastoma, hepatocholangiocarcinoma (females), eosinophilic focus (males), oval cell hyperplasia, and bile duct hyperplasia (females).
Glandular Stomach: malignant neuroendocrine tumors were observed in two 150 mg/kg male mice; one male in this group had a carcinoma. Lesions coexisted and were confined to the fundic region of the glandular stomach, affecting both the more superficial foveolar epithelium of the gastric pits and the deeper epithelium of the gastric glands.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

Under the conditions of this study, there was clear evidence of carcinogenic activity of methyleugenol in male and female B6C3F1 mice based on the increased incidences of liver neoplasms. Neuroendocrine tumors of the glandular stomach in male mice were also considered related to methyleugenol administration.

Key result

Dose descriptor:

LOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Summary of the 2-Year Carcinogenesis study of Methyleugenol

	Male B6C3F1 Mice	Female B6C3F1 Mice
Doses administered in methylcellulose by gavage	0, 37, 75, or 150 mg/kg	0, 37, 75, or 150 mg/kg
Body weights	Dosed groups less than the vehicle control group	Dosed groups less than the vehicle control group
Survival rates	38/49, 36/50, 37/50, 35/50	31/50, 18/50, 18/50, 2/50
Nonneoplastic effects	Liver: eosinophilic foci (10/49, 20/50, 25/50, 19/50); oval cell hyperplasia (0/49, 8/50, 27/50, 46/50); hepatocyte hypertrophy (0/49, 1/50, 7/50, 46/50) Glandular stomach: atrophy (0/49, 3/48, 35/49, 45/50); hyperplasia (0/49, 1/48, 15/49, 20/50); ectasia (13/49, 25/48, 40/49, 49/50)	Liver: oval cell hyperplasia (0/50, 46/50, 36/49, 38/50); hepatocyte hypertrophy (0/50, 10/50, 7/49, 23/50); hepatocyte necrosis (5/50, 9/50, 16/49, 17/50); hematopoietic cell proliferation (4/50, 14/50, 23/49, 24/50); bile duct hyperplasia (1/50, 1/50, 11/49, 9/50); hemosiderin pigmentation (0/50, 11/50, 24/49, 19/50) Glandular stomach: atrophy (0/45, 0/49, 10/46, 10/45); ectasia (14/45, 33/49, 31/46, 38/45)
Neoplastic effects	Liver: hepatocellular adenoma (26/49, 43/50, 38/50, 39/50); hepatocellular carcinoma (10/49, 20/50, 19/50, 9/50); hepatocellular adenoma or carcinoma (31/49, 47/50, 46/50, 40/50); hepatoblastoma (0/49, 0/50, 1/50, 3/50) Glandular stomach: malignant neuroendocrine tumor (0/49, 0/48, 0/49, 2/50)	Liver: hepatocellular adenoma (20/50, 48/ 46/49, 41/50); hepatocellular carcin (7/50, 37/50, 47/49, 47/50); hepatocellula adenoma or carcinom (25/50, 50/50, 49/49, 49/50); hepatoblasto (0/50, 6/50, 11/49, 15/50); hepatocholangiocarci a (0/50, 0/50, 0/49, 2/50)
Uncertain findings
Level of evidence of carcinogenic activity	Clear evidence	Clear evidence

Table 2: Survival of Mice in the 2-Year Gavage Study of Methyleugenol

	Vehicle Control	37 mg/kg	75 mg/kg	150 mg/kg
Male
Animals initially in study	50	50	50	50
Accidental deaths^a	0	0	1	2
Missing ^a	1	0	0	0
Moribund	5	6	5	9
Natural deaths	6	8	7	4
Animals surviving to study termination	38	36	37	35
Percent probability of survival at end of study^b	78	72	76	73
Mean survival (days)^c	690	705	679	686
Survival analysis^d	P=0.815	P=0.825	P=1.000	P=0.829
Female
Animals initially in study	50	50	50	50
Accidental deaths	0	1	1	0
Moribund	5	7	8	13
Natural deaths	14	24	23	35
Animals surviving to study termination	31	18	18e	2
Percent probability of survival at end of study	62	38	37	4
Mean survival (days)	696	662	664	637
Survival analysis	P<0.001	P=0.009	P=0.013	P<0.001

a Censored from survival analyses

b Kaplan-Meier determinations

c Mean of all deaths (uncensored, censored, and terminal sacrifice)

d The result of the life table trend test (Tarone, 1975) is in the vehicle control column [the 300 mg/kg (stop-exposure) group was excluded from the trend test], and the results of the life table pairwise comparisons (Cox, 1972) with the vehicle controls are in the dosed group columns. A lower mortality in a dose group is indicated by N.

e Includes two animals that died during the last week of the study

Table 3: Mean Body Weights of Male Mice in the 2-Year Gavage Study of Methyleugenol

Weeks on Study	Vehicle Control	37 mg/kg		75 mg/kg		150 mg/kg
	Av. Wt. (g)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)
Mean for weeks
1-13	31.0	31.1	100	30.6	99	30.4	98
14-52	48.6	47.9	99	46.4	96	45.5	94
53-101	52.6	50.3	96	48.6	92	48.7	93

Table 4: Mean Body Weights of Female Mice in the 2-Year Gavage Study of Methyleugenol

Weeks on Study	Vehicle Control	37 mg/kg		75 mg/kg		150 mg/kg
	Av. Wt. (g)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)	Av. Wt. (g)	Wt. (% of controls)
Mean for weeks
1-13	24.6	24.4	100	24.1	98	23.3	96
14-52	45.5	43.2	95	40.7	90	35.6	79
53-101	59.9	46.9	79	40.4	68	36.5	61

Conclusions:

Under the conditions of this study, there was clear evidence of carcinogenic activity of methyleugenol in male and female B6C3F1 mice.

Executive summary:

A 2-year carcinogenicity study was conducted to characterize the toxic responses in mice orally exposed to methyleugenol following a method comparable to OECD TG 451 and in compliance with GLP. Groups of 50 male and 50 female F344/N rats received methyleugenol in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg, 5 days per week for 105 weeks. Cage side observations, clinical findings and body weight evolution were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. Survival of all dosed groups of male mice was similar to that of the vehicle controls. Survival of dosed groups of females was significantly less. Mean body weights of dosed mice were generally less than those of the vehicle controls throughout the study. Methyleugenol administration caused significant increases in the incidences of nonneoplastic lesions of the liver and glandular stomach of both male and female mice. Increased incidences of liver neoplasms were observed in both male and female mice. Neuroendocrine tumors of the glandular stomach in male mice were also considered related to methyleugenol administration. Based on these results, methyleugenol is suspected of causing cancer through oral exposure.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

37 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Two experimental key studies are available with a Klimisch score of 2.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available results, methyleugenol is suspected of causing cancer and thus it should be classified as carcinogen Cat. 2, H351 in accordance with CLP Regulation (EC) no 1272/2008.

Additional information

Key study (2 year-study in rats): A 2-year carcinogenicity study was conducted to characterize the toxic responses in rats orally exposed to methyleugenol following a method comparable to OECD TG 451and in compliance with GLP. Groups of 50 male and 50 female F344/N rats received methyleugenol in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg, 5 days per week for 105 weeks; groups of 60 male and 60 female rats received the 0.5% methylcellulose vehicle only. Stop-exposure groups of 60 male and 60 female rats received 300 mg/kg in 0.5% methylcellulose by gavage for 52 weeks followed by just the 0.5% methylcellulose vehicle for the remaining 53 weeks of the study. Cage side observations, clinical findings and body weight evolution were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. All 150 and 300 mg/kg males died before the end of the study, and survival of 150 mg/kg females was slightly less than that of the vehicle controls. Mean body weights of all dosed groups of rats were less than those of the vehicle controls throughout most of the study. Methyleugenol administration caused significant increases in the incidences of nonneoplastic lesions of the liver and glandular stomach of both male and female rats. Increased incidences of liver neoplasms and neuroendocrine tumors of the glandular stomach were observed in both male and female rats. Increased incidences of kidney neoplasms, malignant mesothelioma, mammary gland fibroadenoma, and subcutaneous fibroma and fibroma or fibrosarcoma (combined) were observed in male rats. A marginal increase in the incidence of squamous cell neoplasms of the forestomach may have been related to methyleugenol administration in female rats. Based on these results, methyleugenol is suspected of causing cancer through oral exposure.

Key study (2 year-study in mice): A 2-year carcinogenicity study was conducted to characterize the toxic responses in mice orally exposed to methyleugenol following a method comparable to OECD TG 451 and in compliance with GLP. Groups of 50 male and 50 female F344/N rats received methyleugenol in 0.5% methylcellulose by gavage at doses of 37, 75, or 150 mg/kg, 5 days per week for 105 weeks. Cage side observations, clinical findings and body weight evolution were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. Survival of all dosed groups of male mice was similar to that of the vehicle controls. Survival of dosed groups of females was significantly less. Mean body weights of dosed mice were generally less than those of the vehicle controls throughout the study. Methyleugenol administration caused significant increases in the incidences of nonneoplastic lesions of the liver and glandular stomach of both male and female mice. Increased incidences of liver neoplasms were observed in both male and female mice. Neuroendocrine tumors of the glandular stomach in male mice were also considered related to methyleugenol administration. Based on these results, methyleugenol is suspected of causing cancer through oral exposure.

Supporting study (18 months-study in mice): Methyleugenol was assayed for hepatocarcinogenicity in B6C3F1 male mice after i.p. injections prior to weaning. The test chemical was dissolved in sterile trioctanoin and injected i.p. in a total dose of 4.75 µmol fractioned in the ratio 1:2:4:12 on days 1, 8, 15, and 22 after birth, respectively. 59 mice were weaned at 4 weeks of age. The mothers were fed the purified diet from the date on which they gave birth until the mice were weaned. The young were maintained on the purified diet from weaning until the termination of the experiment. The livers of 23 mice were examined for hepatocarcinogenicity at 13 months by laparotomy. All mice (including those examined by laparotomy) were examined on autopsy at 13 to 18 months. The average number of hepatic tumors induced per mouse was 1.5 at 13 months and 3.2 for the mice examined at autopsy at 13 to 18 months. The solvent-treated and uninjected controls showed average multiplicities of 0.1 to 0.2 and 0.5 tumor/liver on laparotomy at 13 months and autopsy, respectively. Thus, under these test conditions, methyleugenol is concluded to be hepatocarcinogenic.