2,6-di-tert-butyl-p-cresol

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: two generation carcinogenicity study with emphasis on hepatocellular changes in F1 generation

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 for cross-reference:

reference to same study

Data source

Reference

Reference 1

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 2

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

Test material information

Constituent 1

Reference substance name:

2,6-di-tert-butyl-p-cresol

EC Number:

204-881-4

EC Name:

2,6-di-tert-butyl-p-cresol

Cas Number:

128-37-0

Molecular formula:

C15H24O

IUPAC Name:

2,6-di-tert-butyl-4-methylphenol

Details on test material:

IUCLID4 Test substance: other TS: purity: 99.96%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 a previous 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.

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

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.

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

Frequency of treatment:

daily (during the period of mating, food pots were removed when male and females were mated)

Details on study schedule:

Beginning of exposure 3 weeks before mating (F0).
exposure period: male: 5 weeks (P); 4 weeks (F1), 6, 11, 16 and 22 months (F1)
female: 8 weeks (F0)
Duration of test: 22 months

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
F0: 0, 25, 100, 500 mg/kg bw/day
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
F1: 0, 25, 100 and 250 mg/kg bw/day
Basis:

No. of animals per sex per dose:

F0: 7 males and 50 females per group (mating: males 6/dose; females 48/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

Positive control:

none

Examinations

Parental animals: Observations and examinations:

Rats treated with BHT at doses of 25, 100, and 500 mg/kg bw and day showed no significant differences in weight gain or food consumption during pregnancy and lactation, compared with untreated control rats; there were no significant differences in overall mating success between rats treated with BHT and control.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

No statistically significant change was seen in the number of foetuses/dams. 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 deaths in affected litters was not influenced by treatment with BHT.

Postmortem examinations (parental animals):

GROSS NECROPSY: Yes, liver, kidney, thyroid, lung, adrenal and body fat (with emphasis on the liver)

Postmortem examinations (offspring):

GROSS NECROPSY: Yes, liver, kidney, thyroid, lung, adrenal and body fat (with emphasis on the liver)

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.

Reproductive indices:

Pregnancy proceeded normally in all groups of animals.There was no alteration in numbers of resorption sites. No statistically significant change was seen in the number of foetuses/dams.

Offspring viability indices:

The number of pups per litter did not differ between dose groups. 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 deaths in affected litters was not influenced by treatment with BHT

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

prior confirmation of pregnancy; males showed no differences in food consumption, but a tendency of decrease of body weight gain in male rats treated with 500 mg/kg body weight and day

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on fertility

Remarks on result:

other: highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Gross pathological findings:

no effects observed

Effect levels (F1)

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: relative liver weight increased at 11 months post-weaning and some liver enzyme induction

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Maternal toxicity, indicated by a significant increase of the liver weight was seen at 500 mg/kg body weight and day.

There were no differences in mating sucess. Pregnancy proceeded normally in all groups. There was no alteration in numbers of resoption sites. No statistically significant change was seen in the number of foetuses/dam. 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 development was retarded in the high dose group.

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

The NOAEL for toxicity to reproduction was assumed to be 500 mg/kg body weight and day; the NOAEL for maternal toxicity was assumed to be 100 mg/kg body weight and day.

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. Maternal toxicity, indicated by a significant increase of the liver weight was seen at 500 mg/kg body weight and day. No differences in mating success were found in treated dams compared to control. In addition, pregnancy proceeded normally in all groups. There was no alteration in numbers of resorption sites and no statistically significant change was seen in the number of fetuses per dam. The number of pups per litter did not differ (CEFIC-EBMA 1994).

According to the findings of this study, no adverse effects on reproduction were found. The NOAEL for toxicity to reproduction was assumed to be 500 mg/kg body weight and day; the NOAEL for maternal toxicity was assumed to be 100 mg/kg body weight and day.