2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1969

Report date:

1969

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

GLP compliance:

no

Remarks:

Prior to GLP requirements

Test material

Test material information

Constituent 1

Reference substance name:

2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

EC Number:

204-279-1

EC Name:

2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol

Cas Number:

118-82-1

Molecular formula:

C29H44O2

IUPAC Name:

2,6-di-tert-butyl-4-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]phenol

Details on test material:

- Name of test material (as cited in study report): Ethyl Antioxidant 702, identified in report as BHD
- Physical state: crystalline cream colored solid with melting point of 154 degrees C
-

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Brookline, Massachusetts
- Age at study initiation: (P) weanling rats, 2 weeks acclimation, 8 weeks on test diets before initial pairings at about 105 days of age; offspring that would be parents in next generations weaned at 21 days and fed on same diets as progenitors
- Fasting period before study: none
- Housing: 4 females or 3 males per cage if not in mating portion of study; 2 females and 1 male during mating segment; 1 pregnant female in individual nesting box during gestation;

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): as libitum
- Acclimation period: initial animals observed 2 weeks prior to assigning to test groups

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 76.2 +/- 2 degrees F
- Humidity (%): 40 to 60 per cent desired
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Corn oil used to carry test article into feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Special preparation of Rockland Mouse and Rat diet containing no more than 1.0% of fat and free of any antioxidant commonly added as stabilizer and preservative to edible fats was the base diet. For the control diet, 1 kg corn oil added to 19 kg of base diet, and the mixture stirred for 20 minutes. This yielded 20 kg of diet (control) containing not more than 6.0% fat, and no antioxidants other than what occured naturally. For test diets, appropriate amount of test article added to corn oil to make 1 kg and this mixture added to 19 kg of base diet.
- Storage temperature of food: base diet received fresh every 2 months and stored under refrigeration until stored

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil used as carrier to introduce test article into diet, and to supply an adequate nutritional fat for a rat diet, as the base food was restricted to no more than 1.0% fat to avoid fat stabilizing antioxidants
- Concentration in vehicle: 300, 1200, 2000, 10,000 or 60,000 mg test article added to corn oil up to 1 kg.

Details on mating procedure:

- M/F ratio per cage: 1 male, 2 females
- Length of cohabitation: until females showed notable weight gain, or 4 weeks
- After 4 weeks of unsuccessful pairing replacement of first male by another male with proven fertility. Unsuccessful males placed with females of established fertility.
- After successful mating each pregnant female was caged (how): Individual nest boxes

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Initial progenitors: 8 weeks prior to first mating, then throughout gestation, and second mating period and gestation
F1, F2, or F3: from weaning if selected to reproduce, then through mating and two gestations

Frequency of treatment:

Initial group: fed control or treated diets for 8 weeks prior to breeding. Thereafter, animals were on appropriate feed for their group from weaning.

Details on study schedule:

- Age at mating of the mated animals in the study:
Initial animals: 105 days of age, 8 weeks on test diets initially. If mated, allowed to deliver, pups nurse for 7 days, then after 1 week allowed a second mating. If neither female in cage obviously pregnant, a second male placed with females after 4 weeks; second mating as described before.
F1 and F2: initially mated at about 3 months of age, had been on test diets with dams while nursing, and then from weaning on. Second mating after pups allowed to nurse for 7 days, and then allowed to mate after 1 week.

Selection of parents from F1 and F2: Pups of 2nd mating were allowed to nurse for 3 weeks (weaning). At that point, the offspring were grouped by sex, and counted within each dosing group. Animals were reduced in number by random selection to 20 females and 10 males to continue as progenitors of the next generation.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
60 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
500 ppm
Basis:
nominal in diet

Doses / concentrations 5

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

20 females, 10 males per dose group for each breeding generation

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Initial parents: At study initiation
Pups: at birth, 4th day for first breeding offspring of each group, at 7th day and weaning for second breeding group of each dose

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 7 postpartum: in the second breeding offspring for each dose group
- If yes: 20 females and 10 males randomly selected; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Those that died or were killed and examined at 4, 7, or 21 days of age
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, ]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

HISTOLOGICAL EXAMINATION OF PUPS:
The pups of the thrid generation (F3a and F3b) had histological examination of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract, and adrenal glands

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera of all animals dying during the test.

Postmortem examinations (offspring):

SACRIFICE
-The first litter of each initial female was sacrificed after suckling for 7 days. The offspring of the second litter were allowed to nurse 3 weeks, and those not selected for parenting were sacrificed. Pups in the first litters of the F1 and F2 females were counted and weighted at 4 days of age; those of the F1 females were then killed and dams remated. Pups in both litters of the F2 females and those in the second litter of the F1 females wee reduced to 10 pups, and allowed to suclke at 21 days of age. Representatives of the F3 generations were killed at 21 days of age.
- The F3 offspring were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were weighed and prepared for microscopic examination. Tissues were fixed in neutral formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin.
Brain, heart, lungs, liver, kidneys, spleen, intestinal tract and adrenal glands

Reproductive indices:

Fertility index: Number of pregnancies/number of matings x 100
Gestation Index: Number of litters with live pups/Number of pregnancies x 100
Live Birth Index: Number alive at birth/ total number delivered x 100 (Based on first observation of the litters)

Offspring viability indices:

Viability Index: Number of Rats alive on 4th Day/ Total number Delivered x 100 (Day 7 for F1 generations)
Lactation index: Number of pups alive on 21st day/ Number continued after Day 4 x 100
Overall mortality among progeny throuhout 21 days of nursing (in %)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

associated with test article administration

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Associated with test article administration

Reproductive performance:

no effects observed

Description (incidence and severity):

associated with test article administration

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Occasional fatalities among procreating animals were attributable to pneumonia and in some instances to dystocia. Throughout 3 genearations of procreators there werre 13 deaths among 180 males and 360 female progenitors. Three of the 10 females died of dystocia. The presence of disease among the animals, including control animals, was indicated by occasional anorexia, loss of body weight and rales in the lungs. It was thought that a dry atmosphere in the initial animal rooms contributed to the illness.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Test article administration to procreating animals had no effect on fertility or on the extent to which they produced live offspring. Exceptional variation in fertility index and in gestation index were attributed to influence of extraneous disease and not related to test article. Fertility of the initial progenitors (including controls) was poor. Subsequent generations had satisfactory performance. The number of pregnanccies resulting from the matings showed no relationship to test article in diet. During the two periods of mating of the initial progenitors, more pregnancies occurred in treated females (3000 ppm in diet) than in controls. The least number of pregnancies in th e(P) rats was in the animals with 60 ppm in the diet. Among the first generation of offspring (F1) the fertility index was lowest in the group fed 3000 ppm, and highest (100%) in the animals fed 500 ppm in the diet. Among the second generation (F2) of progeny, fertility was slightly low in both periods of mating in animals at 15 ppm in the diet. It was also slightly low in the first period, but not the second period, of mating of the rats fed 100 ppm. As there was no consistent pattern of effect, the differences in fertility were not thought to be attributalbe to test article administration.
Of the 180 male progenitors in the whole test, only 7 failed to sire litters; five of those were inital progenitors (3 at 60 ppm, 2 at 500 ppm). One male of the F1 generation on the 3000 ppm diet and one male of the F2 generation on the 15 ppm level also failed to sire offspring. These failures were attributed to sporadic disease unrelated to the test article.
The gestation index was lowest during the second period of mating of the original progenitors in the control group. It was consistently high among females of the 500 and 3000 ppm diet groups. Of the 60 females of the 3 generations on test, there were no differences attributable to test article in the numbers that produced 0, 1, or 2 litters when the means were compared by chi-square.

Effect levels (P0)

Dose descriptor:

NOEC

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: Parental, F1, F2, F3 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

associated with test article

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

associated with test article

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No remarkable effect on weights at birth, day 4 (7) or when weaned at 21 days

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No anatomic anomaly, structural abnormality in the 5400 progeny examined. No gross pathology found in F2 and F3 offspring

Histopathological findings:

no effects observed

Description (incidence and severity):

No histopathology in tieeuse of third generation offspring examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

VIABILITY (OFFSPRING): Probability of survival at birth, and during first days of life was generally poor among th efirst generatin progeny ranging from 45.9 to 73% without regard to test article in the diets of the parents. The viability index improved in the second generation ranging from 89.4 to 97.4%, and was normal in the third generation except for the offspring of parental group receiving 60 ppm test article in diet; viability in that group was 84.9%, The poor life expectancy of the progeny was attributed in every instance to the influence of extraneous disease, and not the test article.
Survivability during nursing period (lactation index) ranged from 47.3 to 91.9% in the second generation and from 66.5 to 94.6% in the third generation; differences were not attributable to test article administered to the lactating females.

CLINICAL SIGNS (OFFSPRING): No remarkable differences in clinical signs were seen in the offspring of treated procreators compared with control pups

BODY WEIGHT (OFFSPRING): Content of test article in the procreating animals had no remarkable effect on body weights of the offspring at birth, at 4 days of age, or when weaned at 21 days.


GROSS PATHOLOGY (OFFSPRING): No gross pathological changes were found in the F2 and F3 offspring.

HISTOPATHOLOGY (OFFSPRING): Histopathological exam of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract and adrenal glands of the third generation of offspring (F3) revealed no pathological alterations.

Effect levels (F1)

Dose descriptor:

NOEC

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: clinical signs; mortality; body weight; gross pathology; organ weights; histopathology; mating index; fertility index; birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; survival index; viability index; lactation ind

Overall reproductive toxicity

Reproductive effects observed:

not specified

Lowest effective dose / conc.:

3 000 ppm

Treatment related:

not specified

Any other information on results incl. tables

The Gestation Index of Successive Generations of Rats on Diets with and without Test Article (Table 1 )

Test article concentration in diet (ppm)	1^stMating Period (%)	2^ndMating Period (%)	Average for Both Matings (%)
Initial Parents (P)
0	100.0	75.0	87.9
15	91.7	89.5	90.3
60	100.0	100.0	100.0
100	100.0	83.3	90.9
500	100.0	93.8	96.8
3000	100.0	93.8	97.0
Generation F1
0	100.0	100.0	100.0
15	100.0	94.7	97.4
60	94.7	100.0	97.4
100	100.0	94.1	97.2
500	100.0	100.0	100.0
3000	100.0	100.0	100.0
Generation F2
0	100.0	100.0	100.0
15	100.0	100.0	100.0
60	100.0	90.0	94.7
100	100.0	100.0	100.0
500	100.0	94.7	97.4
3000	100.0	94.4	97.3

Gestation Index:   Number of litters with live pups/Number of pregnancies x 100

Groupings of Females Fed Each Diet and the Number of Litters They Produced (Table2)

Test Article Concentration in the Diet (ppm)	Number of females which produced the indicated number of litters
Number of Litters	0	1	2
0	2	8	50
15	5	11	44
60	6	14	40
100	3	14	43
500	2	10	48
3000	4	11	45
No Test Article	2	8	50
Test Article Diets	20	60	220

Diets with and without Test Article (Viability Index) (Table 3)

Test article concentration in diet (ppm)	1^stMating Period (%)	2^ndMating Period (%)	Average for Both Matings (%)
Generation F1
0	81.3	61.6	73.1
15	83.7	36.5	55.7
60	81.9	64.9	73.1
100	68.3	20.8	45.9
500	83.3	29.4	56.0
3000	74.7	31.5	56.2
Generation F2
0	96.3	98.5	97.4
15	96.3	90.4	93.3
60	92.3	95.7	94.1
100	99.4	80.3	89.4
500	98.6	88.8	93.4
3000	98.9	92.8	96.2
Generation F3
0	96.4	91.5	93.8
15	97.0	96.4	96.7
60	88.4	81.5	84.9
100	96.5	98.0	97.3
500	98.9	92.6	95.6
3000	98.8	94.9	96.6

* Seventh Day for Generation F1  

Viability Index: Number of Rats alive on 4^thDay/ Total number Delivered x 100

Percentage Number of Animals Alive at Birth (Live birth Index) (Table 4)

Test article concentration in diet (ppm)	1^stMating Period (%)	2^ndMating Period (%)	Average for Both Matings (%)
Generation F1
0	93.6	81.2	87.4
15	96.3	80.7	88.5
60	98.1	98.2	98.2
100	94.6	84.6	89.6
500	98.5	91.2	94.8
3000	98.8	92.3	95.6
Generation F2
0	97.9	100.0	99.0
15	97.2	96.1	96.6
60	94.2	97.0	95.6
100	100.0	98.9	99.4
500	99.0	98.3	98.6
3000	100.0	100.0	100.0
Generation F3
0	99.5	98.6	99.0
15	99.5	100.0	99.8
60	97.0	97.6	97.3
100	100.0	100.0	100.0
500	99.5	99.0	99.2
3000	100.0	97.2	98.6

Live Birth Index: Number alive at birth/ total number delivered x 100

(Based on first observation of the litters)

Applicant's summary and conclusion

Conclusions:

The test substance was administered in diet at 0, 15, 60, 100, 500, and 3000 ppm (nominal) to 3 successive generations of breeding rats. Over the course of the study, animals were affected by environmental conditions and extraneous disease that were unrelated to test article in the diet. However, it can be concluded from this study that the substance in the diet up to levels of 3000 ppm did not affect fertilityof rats, viability of offspring, or caused abnormalities in the offspring under the conditions of this 3-generation study

Executive summary:

Diets containing the test substance in the nominal amounts of 0, 15, 60, 100, 500, and 3000 ppm were fed to 3 successive generations of breeding rats. Over the course of the study, animals were affected by environmental conditions and extraneous disease that were unrelated to test article in the diet. However, the data warrant the following conclusions:

1. Occasional fatalities among the breeding animals were attributable to respiratory disease, and in some cases dystocia.

2. The content of test article in the diet had no effect on fertility of the breeding animals, or on the extent to which they produced living offspring. Exceptional variations in fertility index and in gestation index were attributed to the influence of extraneous disease unrelated to test article concentration in diet.

3. The probability of survival at birth and during the first days of life, the viability index, was generally poor among the first generation ranging from 45.9 to 73 per cent without regard to test article concentration in the diet fed to the parents. The viability index improved in the second generation ranging from 89.4 to 97.4% and was normal in the third generation except for the group fed a test article diet containing 60 ppm. At that concentration level, viability was 84.9%. The poor life expectancy of offspring in the various groups was attributed to extraneous factors and not test article concentration in the diet.

4. The survival during the time the pups were nursed (the lactation index) ranged from 47.3% to 91.9% in the second generation, and from 66.5 to 94.6% in the third generation. The variation was not attributed to test article concentration in the diet.

5. The content of test article in the diets of the breeding animals exerted no remarkable effect upon the body weights of the offspring at birth, at 4 days of age, or when weaned at 21 days of age.

6. No anatomical anomaly or structural abnormality was found among 5400 rats that were offspring of animals on diets containing test article.

7. No gross pathological changes were found in the F2 and F3 offspring. Histological examination of the brain, heart, lungs, kidneys, liver, spleen, intestinal tract, and adrenal glands of the third generation of offspring revealed no pathologic alterations.