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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-study, comparable to guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Hepatic and associated response of rats to pregnency, lactation and simultaneous treatment with butylated hydroxytoluene
Author:
McFarlane, M.; Price, S., C.; Cottrell, S.; Grasso,P.; Bremmer, J., N.; Bomhard, E., M.; Hinton, R., H.
Year:
1997
Bibliographic source:
Food and Chemical Toxicology, 35, 753-767
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Principles of method if other than guideline:
other: two generation carcinogenicity study with emphasis on hepatocellular changes in F1 generation
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TS: purity: 99.96%

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Male Wistar albinio rats weighing approx. 200 g and female Wistar albino rats weighing approx. 60 g were obtained from Bantin and Kingman (Hull, UK) (F0 generation). The animals were housed in polypropylene cages; room temperature maintained at 20 +/- 3°C, humidity: 30-70%, 12 h light/dark cycle; male rats were housed singly, females in groups of seven or eight.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Details on exposure:
Diet mixes were prepared every two weeks during the initial phase of the study (i.e. F0 or breeding phase) and stored at 4°C as it has been shown in aprevious study that the BHT in the diet was not stable for long periods in the food pots at room temperature. The rats were reweighed prior to the diet mixing, and the concentration of BHT in the diet readjusted to maintain the required intake, based on measured diet consumption and from historical data on the growth curve of this strain of rats. The concentration of BHT in each batch of diet at each dose level was analysed before administration of that batch to the F0 test animals. For the F1 generation diet mixes were prepared weekly for the first four weeks, and thereafter at fortnightly intervals and stored at 4°C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of BHT in diet were tested with HPLC. The concentration of BHT in each batch of diet at each dose level was analysed beforeadministration of that batch to the F0 test animals. For the F1 generation diet mix were prepared weekly for the first 4 weeks, and thereafter at fortnightly intervals and stored at 4°C. Analysis of the diet was carried out once every 3 months post-weaning. This diet was analysed on the day of mixing (Day 0). Samples of diet were then taken 14 days later and frozen at -20°C. These samples were then analysed with another batch of diet (day 0) prepared three months later. However, a sample of every diet batch prepared was taken and stored at -20°C for possible analysis.
Details on mating procedure:
mating exposure period for males (6/dose) and females (48/dose): 2 weeks; M/F ratio per cage: 1/8; lengh of cohabitation: 15 hours/day
Duration of treatment / exposure:
exposure period: male: 5 weeks (F0); 4 weeks (F1), 6, 11, 16 and 22 months (F1)
female: 8 weeks (F0)
Premating exposure period (males): 3 weeks
Premating exposure period (females): 3 weeks
Frequency of treatment:
daily
Duration of test:
F1: up to 22 months
No. of animals per sex per dose:
F0: 7 males and 50 females per group (mating: 6 males and 48 females per dose)
F1: 5-20 males (number of animals examined at different time points)
Control animals:
yes, plain diet
Details on study design:
dose ranging study was done for dose selection

Examinations

Maternal examinations:
BODY WEIGHT: Yes
Fetal examinations:
External examinations: Yes
Statistics:
Student's t-test, analysis of variance and regression analysis. For pathological findings the trend for analysis as recommended by Peto et al (1980) IARC, Supplement, was employed.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
dams receiving 500 mg BHT/kg body weight and day showed a significant increase in liver weight

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: no adverse effects on developmental toxicity
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: reduced body weight of pups at weaning and retarded development

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

There were no differences in food consumtion or body weight gain between female control rats and female rats fed BHT-contained diet. In contrast, for the male animals, although food consumption was simular, there was a tendency for the body weight to be low, i.e. reduced body weight gain, in male rats treated with 500 mg/kg body weight and day BHT then in the lower dose groups and control.

There was no evidence of ill health in either male or female rats prior confirmation of pregnancy. There were no differences in mating success. Pregnancy proceeded normally in all groups. The weight of the dams on the day of birth of pups gave no evidence for any differences between control and BHT-treated animals. The dams were in good health prior to autopsy and no abnormalities were observed at autopsy with the exception of one dam treated with 25 mg/kg body weight/day BHT (congenitally deformed leg) and one treated with 500 mg/kg body weight/day BHT (darker liver).

The liver weight of the dams treated with 500 mg/kg body weight/day BHT was statistically increased compared to control when expressed as absolute weight, relative to the weight of the pregnant dam or relative to the weight of the dam less the weight of the litter. Livers from 4 out of 5 dams treated with 500 mg/kg body weight/day and 1 out of 5 dams treated with 25 mg/kg body weight/day BHT showed mild centrilobular enlargement and eosinophilia, which are commonly associated with induction of mixed function oxidase activity. No changes were seen in rats treated with either 100 mg/kg body weight/day BHT or the remaining 4 rats treated with 25 mg/kg body weight/day BHT when compared to control.

There was no alteration in numbers of resorption sites. No statistically significant change was seen in the number of foetuses/dam.

No effects was seen in the body weight, liver weight or liver body weight ratio in foetuses at the estimated 20th day of gestation.

The number of pups per litter did not differ. There was a trend to an increase in the number of pups found dead or dying soon after birth with increase in dose but the actual number of death in affected litters was not influenced by treatment with BHT.

The total litter weight was significantly decreased for dams treated with the high dose of BHT. The weight gain of pups from dams receiving the highest dose of BHT was consistently less than that of control pups or pups of dams receiving lower dose of BHT. The body weight gain of the pups of the highest dose group at weaning was 14 % lower than that of the control (46.2 g vs. 53.8 g). The development was retarded in the high dose group.

The NOAEL for maternal toxicity was assumed to be 100 mg/kg body weight and day. The NOAEL for pups was assessed to be 100 mg/kg body weight and day.

The pathology findings (F0 and F1, including liver-biochemistry, organ weights, gross and microscopic evaluation are presented in chapter "Repeated Dose Toxicity and Carcinogenicity".

Applicant's summary and conclusion

Executive summary:
The results of this two-generation carcinogenicity study, revealed no adverse effects on reproductive performance of the F0 generation fed with BHT at dietary levels of 25, 100 or 500 mg/kg body weight. However, dams receiving 500 mg/kg body weight and day showed a significant increase in liver weight. The NOAEL for maternal toxicity was assumed to be 100 mg/kg body weight and day. No adverse effects on fetal development were observed, but there was a significant reduction in body weight gain of pups nursed by dams on the highest dose group (500 mg/kg body weight). In addition, the development was retarded in pups of the high dose group. The NOAEL for pups was assessed to be 100 mg/kg body weight and day and the NOAEL for maternal toxicity was assumed to be 100 mg/kg body weight and day (CEFIC-EBMA 1994).