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Toxicological information

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: Study followed acceptable scientific practice for the time period.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1970
Report date:
1970

Materials and methods

Principles of method if other than guideline:
Artificially inseminated rabbits received test article in gelatin capsules, or empty capsules if in control group. Animals observed daily, and were Caesarian section performed on Day 28, 29 or 30 of pregnancy. Uterine and ovarian observations were made of implantation sites, corpora lutea, and fetuses (live and dead) counted, measured and weighed. Fetuses were examined for soft tissue development, and stained and examined for skeletal development.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
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:
2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol
Details on test material:
- Name of test material (as cited in study report): EY 2333
- Physical state: Fine white powder

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rowmar rabbitry
- Age at study initiation: Adults of proven fertility
- Diet (e.g. ad libitum): Purina Chow ad libitum
- Water (e.g. ad libitum): Ad libitum

IN-LIFE DATES: From: Insemination April 7, 1970. To: Caesarean section May 7, 1970

Administration / exposure

Route of administration:
oral: capsule
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): No vehicle, powder in gelatin capsules
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: artificial insemination after stimulation of ovulation by administration of Pituitary Luteinizing Hormone
- Verification of same strain and source of both sexes: yes
Duration of treatment / exposure:
Dose administered once daily from day 6 to day 18 of pregnancy
Frequency of treatment:
Once daily
Duration of test:
Caesarian section on day 28, 29 or 30 of gestation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg/kg body weight
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
15 females bred per group
Control animals:
yes, sham-exposed

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: mortality, appearance, behavior

BODY WEIGHT: Yes
- Time schedule for examinations: study intiation, weekly, and termination

FOOD CONSUMPTION Yes, recorded daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #28, 29, or 30
- Organs examined: Uterus, ovaries in particular

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes, right, left
- Number of implantations: Yes right horn, left horn
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number pups, and location in horns
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Indices:
Ratio of implantation sites/ corpora lutea

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: that could be attributable to test article

Details on maternal toxic effects:
Maternal data: One death occurred in a control female, and one in low dose treated females. Both animals that died were found on Day 6 following dosing; pregnancy could not be determined. In those animals which survived until study termination, three control females had aborted.
One low dose animal was not pregnant, although one corpus luteum was found. Six high level females were not pregnant. Two of the high dose non-pregnant animals had three corpora lutea each. Animals that were pregnant and did not abort were comparable in body weights and food consumption.
Uterine/Caesarian data: Ratio data included those pregnant animals in which CL in both ovaries were determined. The ratio between implantation sites and number of corpora lutea observed was comparable between control and treatment groups.
There were no meaningful differences between control and test groups in the number, and placement of implantation and resorption sites, in the number, weight, and length of live and dead fetuses, or in incubation survival. The number of dead fetuses was slightly elevated in the control and high levels. In the control group, 10 litters were evaluated, and three litters had resorption sites, and 2 had dead fetuses. In the 15 mg/kg/day group, 13 litters were evaluated; 5 had resorption sites and 5 litters had dead fetuses. In the 150 mg/kg/day group, 9 litters were evaluated; 4 litters had resorption sites and 5 had dead fetuses.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: that could be attributed to test article

Details on embryotoxic / teratogenic effects:
External and Visceral Data for Fetuses: Three low level and 5 high level fetuses found dead at Caeserian section were too small to weigh or measure. Bruising was noted in four dead control pups and one live pup from one litter that were delivered normally. Ten of the dead aborted control fetuses were macerated or mutilated. One live low level fetus had all the first digits of the forepaw missing.
Skeletal data: Evaluations of the skull, ribs, sternum, vertebrae, pelvic girdle, long bones, fore and hind paws of the animals were made. The fetuses from the three aborted control litters, three dead low level fetuses, and six dead high dose level fetuses were either too small or damaged to be processed for skeletal evaluation. The fetuses delivered by Caesarian section and those delivered naturally wee stained and cleared. Single ribs, ribs described as small, and ribs described as “floating” in appearance or unattached from the vertebral column were located at the 20th vertebrae in all animals in which they were observed. Rudimentary bone structures noted on the left side of the vertebral column were observed at the 20th vertebra in two control and one high level fetus. One low level fetus had one nonossified portion of the left parietal bone and two in the right. Several (numbers 1-4) sternabrae were irregular in shape in one high level fetus; an extra ossification center was located on the first sternebra in one control fetus. All of the first digits in both forepaws were missing with no signs of any structure in one low level fetus.
There were no meaningful differences in the developmental indices of the test groups as compared to control. No trends towards lesser or greater skeletal development were observed.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Uterine and Litter Data

Group

(No. Pregnant/died/

aborted)

Implant sites (mean)

Resorption sites (mean)

Live Fetuses

(mean)

Dead Fetuses (mean)

Weight/ Length

Live Fetuses (mean) grams/cm

Weight/

Length Dead fetuses

(mean) grams/cm

Incubation Survival (Percent)

Control (14/1/3)

9.1

0.3

7.6

1.2

36.8 /8.5

18.5/6.3

87.5

15 mg/kg

(13/1/0)

9.3

0.7

8.2

0.4

38.6/8.8

19.3/6.8

77.6

150 mg/kg

(9/0/0)

10.8

0.7

8.8

1.3

38.2/8.8

15.0/5.3

81.8

Skeletal Examination Data

Skeletal Effect

Control

15 mg/kg group

150 mg/kg group

Skull

  Closure Grade 0

0

0

0

                          1

0

0

0

                          2

0

0

3

                          3

2

4

3

                          4

94

105

79

  Nonossified Parts, Parietal Bone

0

1

0

Ribs

  Pairs 12

79

91

67

  Pairs 13

15

18

18

  Pairs 14

1

0

0

  Single

10

11

10

  Small

16

24

22

  Floating

6

7

8

  Rudimentary

2

0

1

Vertebrae - caudal

 Nonossified Centra

                          2

0

1

0

                        13

0

0

1

 Nonossified Dorsal Arches

                          2

0

1

0

                        13

0

0

1

Sternum Ossification Centers

  Absent          1st

0

0

1

                        2nd

0

0

2

                        3rd

0

0

1

                        4th

0

0

3

                        5th

49

46

36

                        6th

8

3

10

  Small            1st

2

1

3

                        2nd

4

3

5

                        3rd

1

1

1

                        4th

3

3

1

                        5th

44

52

35

                        6th

19

34

20

  Split              1st

0

0

0

                        2nd

1

0

0

                        3rd

1

0

0

                        4th

1

0

0

                        5th

5

3

3

                        6th

5

2

0

  Irregular

0

0

1

  Extra center

1

0

0

Pelvis Ossification Centers Absent

  Right Pubis

0

3

5

  Left Pubis

0

3

5

Forepaws Ossification Centers

  Total Number Absent

    Carpus             4

96

109

85

    Metacarpus   1-2

19

14

17

                          3-4

0

1

2

                        5-10

0

0

2

    Phalanges   1-15

40

23

25

                      16-20

0

1

3

                      21-28

0

0

1

Hindpaws Ossification Centers

  Total Number Absent

    Tarsus                2

1

4

2

                               4

0

0

5

    Metatarsus       1-2

0

0

1

                              3-4

0

0

0

                             5-10

0

0

2

    Phalanges        0-15

12

11

13

                            16-20

0

0

2

                            21-24

0

0

1

Applicant's summary and conclusion

Conclusions:
In a rabbit teratology study, where female rabbits were dosed by capsule on days 6 through day 18 of pregnancy, there were no effects that could be related to test article as to maternal mortality.  Appearance, behavior, body weight gain, and food consumption were comparable among control and test animals. All uterine, litter, and Caesarian data were also comparable. There was no indication of a treatment related effect in fetal external appearance, visceral anatomy, development and skeletal structure of the test offspring compared to control.
Executive summary:

Forty-five female albino rabbits were bred by artificial insemination after stimulation of ovulation by administration of Pituitary Luteinizing Hormone. Fifteen animals were assigned to each test or control group. Test article was administered orally by capsule to female rabbits from day 6 to day 18 of pregnancy. Test article doses were either 15 or 150 mg/kg body weight, and control animals received empty gelatin capsules. Pregnancy was observed in 36 of the 45 animals bred (14 control, 13 low dose group, 9 high dose group). One control and one low dose female died during the study before pregnancy could be determined. Abortion was noted in 3 control animals. One control animal delivered naturally; all other control and test litters were delivered by Caesarian section.  Appearance, behavior, body weight gain, and food consumption were comparable among control and test animals. All uterine, litter, and Caesarian data were also comparable. There was no indication of a treatment related effect in fetal external appearance, visceral anatomy, development and skeletal structure of the test offspring compared to control.