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Repeated dose toxicity: oral

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2000
Reference Type:
publication
Title:
14-Week toxicity and cell proliferation of methyleugenol administered by gavage to F344 rats and B6C3F1 mice
Author:
Abdo K M
Year:
2001
Bibliographic source:
Food Chem Toxicol 39, pp. 303-16

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
4-allylveratrole
EC Number:
202-223-0
EC Name:
4-allylveratrole
Cas Number:
93-15-2
Molecular formula:
C11H14O2
IUPAC Name:
1,2-dimethoxy-4-(prop-2-en-1-yl)benzene
Test material form:
liquid

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

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

 

 

Mean Body Weightb(g)

 

Dose (mg/kg)

Survivala

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

 

 

 

 

 

 

 

Livera

10

10

10

10

10

9

10

  Cytologic Alterationb

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

Applicant's summary and conclusion

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.