Ethylene bis[3,3-bis(3-tert-butyl-4-hydroxyphenyl)butyrate]

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Dec 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed and reported non-guideline study with scientific sound design and sufficient reporting.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Rats were fed diets containing the test item at different levels and mated. The litters were reared and observations were made on fertility of females, number of young born per litter, sex ratio, grossly visible abnormalities, mortality, body weights and resorption percentage.

GLP compliance:

no

Remarks:

performed before GLP guidelines

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DTB-glycolester

IUPAC Name:

DTB-glycolester

Constituent 2

Reference substance name:

Bis-(3,3-di(4-oxy-3 tert. butyl-phenyl)butane acid)-glycolester

IUPAC Name:

Bis-(3,3-di(4-oxy-3 tert. butyl-phenyl)butane acid)-glycolester

Constituent 3

Reference substance name:

Hostanox O 3

IUPAC Name:

Hostanox O 3

Test material form:

solid: crystalline

Details on test material:

white crystalline material

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: (P) Males: 268 g; Females: 179 g
- Housing: in groups of five in screen-bottomed, stainless steel cages
- Diet (e.g. ad libitum): CIVO basal diet (containing 29.7% yellow maize, 36% whole wheat, 11% defatted soy-bean mael, 4% meat scraps, 7% fish meal, dried whey, brewer's yeat, grass meal, soy-bean oil, vitamin preparations, trace mineralized salt, steamed bone meal), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25°C
- Humidity (%): 50%
- lighting 12 h daily

3 weeks acclimatisation

IN-LIFE DATES: From: Aug. 1978 To: May 1981 (end of subsequent chronic toxicity study)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: CIVO basal diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
The test diets were normally prepared in batches once every 3 to 4 weeks.
The test material was thoroughly mixed into CIVO basal diet by means of a mechanical blender at levels of 0 (control), 0.16, 0.4 or 1.0%. The diets were stored in an unheated room at ambient temperatur

Details on mating procedure:

After the pre-mating period (30 days) the rats were mated within their diet group. Each male was housed with two females in a cage for one week. At week 2 and 3 of the mating period each male rat was transferred to another "mating" cage within the same diet group. So, three different males were available for each dam. After a mating period of three weeks, the females were caged individually, until their litters had been weaned.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

Males: 7 weeks (4 weeks pre-mating, 3 weeks mating)
Females: 13 weeks (4 weeks pre-mating, 3 weeks mating, 3 weeks gestation, 3 weeks lactation)

Frequency of treatment:

continuously

Details on study schedule:

- Age at mating of the mated animals in the study: 15 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.16%
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.4%
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.0%
Basis:
nominal in diet

No. of animals per sex per dose:

15 males/dose
30 females/dose

Control animals:

yes, plain diet

Details on study design:

no data

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
In the pre-mating period of 30 days food intake was recorded at weekly interval.

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

no data

Litter observations:

Records were made of the number of pups in each litter, sex ratio at birth and the weight of the litter at day 1,4, 14 and 21 of lactation. Litters containing more than 8 siblings were randomly reduced to 8 on day 1 of lactation, in order to equalize the stress of lactation among the dams.

Postmortem examinations (parental animals):

After weaning their young, the mothers were sacrificed and the implantation sites in the uterus were counted after staining with ammonium sulfide solution. The males were discarded.

Postmortem examinations (offspring):

n.a., since the offspring was used a subsequent combined chronic toxcity/carcinogenicity study (please refer to study report no. R 6693; IUCLID Chapter 7.5.1; WoE_130 week oral toxicity (diet)_rat_TNO_1982)

Statistics:

All data were evaluated by means of the Student t-test.

Reproductive indices:

- female fertility index = (number of pregnant females/number of females placed with males) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Offspring viability indices:

- Sex ratio at birth = no. of males alive/no. of females alive
- Viability Index at day 1 = (No. of pups born alive/total no. of pups born) x 100
- Viability Index at day 4 = (No. of pups alive days 4/total no. of pups alive day 1) x 100
- Lactation Index = (No. of pups alive day 21/no. of pups alive on dy 4) x 100

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

Description (incidence and severity):

slightly reduced in males at mid- and high dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly reduced in males at mid- and high dose

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: slightly reduced in males at mid- and high dose

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)

- General appearance, growth, and food intake of parent rats:
No abnormalities of condition or behaviour were observed in any of the groups during the pre-mating, mating or lactation period. At 0.4 and 1% dose level body weights were relatively low in males, while in females the figures of the various groups were very similar. Food intake tended to be decreased in all test groups, but the differencs with the control group were very small.

- Fertility:
All females of the control group and of the 0.16 and 0.4% dose group casted a litter while in the 1.0% dose group only 1 out of 30 females was not fertile. The mean litter size at birth showed some variation amongst the groups, but there were no indications of an adverse effect of the test substance. The resorption quotient of the various groups did not reveal any embryo-toxic properties of the test substance. None of the other parameters examined was affected by the feeding of the test item.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 other: % in diet

Sex:

male/female

Basis for effect level:

other: only slightly reduced body weight in males of high dose group observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced in high dose group

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

The total number of pups born dead in each group was only small, resulting in a high viability index at day 1. The sex ratio (male/female) of the pups at birth tended to decrease with increasing levels of DTB-glycolester in the diet. Mortality of the offspring during lactation, as expressed by the viability index at day 4 and the lactation index, was low and was not adversely affected by the test substance. The mean pup weight at day 1, 4 and 14 was comparable in the various groups, but at day 21 body weights in the high dose group were lower than in controls.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 other: % in diet

Sex:

male/female

Basis for effect level:

other: reduced body weight in high dose group

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

HostanoxO 3 was studied for its reprodcutive toxic/teratogenic properties in a one-generation combined chronic toxicity study in male and female Wistar rats. The NOAEL was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested) in diet. None of the fertility/viability parameters examined was adversely affected by the feeding of the test substance. Slightly reduced body weight was observed in males and pups of the high dose group.

Executive summary:

The feeding of DTB-glycolester at dietary levels of 0, 0.16, 0.4 or 1% (corresponding to 0, 70, 200 and 500 mg/kg bw/d considering a diet conversion factor (ppm to mg/kg bw/d) of 20) to rats resulted in slight growth depression and relatively low food intake in male parent rats of the mid and high dose group. The only change observed in the litters consisted of slight growth retardation of the pups in the top dose group during the final stage of the lactation period. The other data on fertility, lactation performance and survival did not reveal any abnormalities.

From the present reproduction study it is therefore concluded that the feeding of the test substance at dietary levels up to 1.0% failed to induce any deleterious effects, other than slight growth depression in parent males at 0.4 and 1.0% and in pups at 1.0%.

The F1 -generation was used for a subsequent chronic toxicity study (130 weeks duration, please refer to IUCLID Chapter 7.5.1). No visceral malformations could be found at the termination of this chronic study and therefore, no teratogenic effects were recorded. Furthermore, the rate of mortality was not affected, the body weight differences of rats of the dose groups with the control animals were smaller than 10% and there were no outstanding differences in food intake observed during the chronic toxicity study performed in the F1 -generation.

The NOAEL was considered to be 1% (corresponding to 500 mg/kg bw/d; highest dose tested) in diet. None of the fertility/viability parameters examined was adversely affected by the feeding of the test substance. Slightly reduced body weight was observed in males and pups of the high dose group.