N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981-05-12)

Principles of method if other than guideline:

examination of dams and progeny in accordance with WHO (WHO 1975) recommendations
(Wld. Hlth. Org. Tech. Rep. Ser. 563, 1975)

GLP compliance:

no

Remarks:

(GLP was not mandatory when study was done)

Limit test:

no

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 2 months (females)
- Weight at study initiation: 200 g (females)
- Housing: 5 per cage(for successfully mated females)
- Diet (e.g. ad libitum): standard diet Nafag No. 890
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 0.5
- Humidity (%): 60 ± 5
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

2 % solution

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): 20 mL/kg bw
- Lot/batch no. (if required): Prod. No. 8348

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation: cohoused
- If cohoused:
- M/F ratio per cage: 1 : 3
- Verification of same strain and source of both sexes: no
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 6- 15 (females only) of gestation

Frequency of treatment:

daily, 7 days/week (females only)

Duration of test:

day 21 of pregnancy

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (during the treatment only)

BODY WEIGHT: Yes
- Time schedule for examinations: daily (during the treatment only)

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: at day 6, 11, 16, 21 of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: following the recommendations of the WHO (WHO 1975), especially ovaries and uterus (mucosa and contents, including amniotic fluid and placentae)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter]
- Skeletal examinations: Yes: [ 2/3 per litter]
- Head examinations: No

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 3000 mg/kg bw dose group the body-weight gain was slightly depressed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was found to be increased at the three doses during the last period of treatment (days 12-15 of pregnancy) at a dose-relationship.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The implantation rates were comparable for all experimental groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The embryolethality rates (resorptions) were not significantly increased when compared with the vehicle control (chi-square test, Yate's correction).

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The average weight of the foetuses were comparable for all experimental groups.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One out of the 278 live foetuses of the 100 mg/kg dose group showed cranioschisis in association with cheilognathoschisis (cleft upper jaw and lip). No malformations were found in both the other dose groups and the vehicle control. The spontaneous malformation rate is derived from a series of untreated controls (cumulative control). By applying the slicing technique pulmonar hypoplasia and anasarca were detected in either the vehicle control or the cumulative control. One foetus of this cumulative control displayed microphthalmia.

Skeletal malformations:

no effects observed

Description (incidence and severity):

The skeletal assessment on the dose groups did not reveal any clearcut deviation from the vehicle control.

Visceral malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981-05-12)

Principles of method if other than guideline:

examination of dams and progeny in accordance with WHO (WHO 1975) recommendations
(Wld. Hlth. Org. Tech. Rep. Ser. 563, 1975)

GLP compliance:

no

Remarks:

GLP was not mandatory when study was done

Limit test:

no

Species:

mouse

Strain:

Tif:MAGf

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 to 6 weeks (females)
- Weight at study initiation: (mean body weight) 30g
- Housing: 5 per cage in macrolon cages
- Diet: Nafag No. 890
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1
- Humidity (%): 60 ± 5
- Photoperiod (hrs dark / hrs light): 12/ 12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

VEHICLE
- Concentration in vehicle: 2 % aqueous solution in sodium carboxymethylcellulose (CMC)
- Amount of vehicle: 0.2 mL/10g bw
- Lot/batch no.: Prod. No 8348

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation: cohoused (overnight)
- If cohoused:
- M/F ratio per cage: 1 : 4
- Proof of pregnancy: vaginal plug referred as day 0 of pregnancy

Duration of treatment / exposure:

day 6-15 of pregnancy

Frequency of treatment:

daily from day 6 to day 15 of pregnancy

Duration of test:

day 18 of pregnancy

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (during the treatment only)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (during the treatment only)

BODY WEIGHT: Yes
- Time schedule for examinations: daily (during the treatment only)

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: on days 5, 10, 15 of pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
- Organs examined: specially ovaries and uterus (mucosa and contents, including amniotic fluid and placentae)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter]
- Skeletal examinations: Yes: [ 2/3 per litter]
- Head examinations: No

Statistics:

chi-square-test, Yate's correction
student's t-test, one-tailed

Historical control data:

Spontaneous data from a series of untreated controls

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight gain was comparable in all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the 100 and 1500 mg/kg bw group food intake was slightly reduced.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The implantation rates were comparable for all groups.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

At the 1500 mg/kg dose the embryolethality rate (resorptions) appeared to be slightly increased; there was, however no significant difference when compared with the vehicle control (chi-square test, Yate's correction, p < 0.01). The percentage of embryonic resorptions is 8.0 % of high dose group vs. 4.2 % for vehicle control and 6.6 % for historical control data.

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): In the 1500 mg/kg dose group the average weight of the live foetuses was slightly but significantly diminished (Student's t test, one-tailed, p < 0.01) 1.15 g vs 1.19 g.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The ratios male/ female were not significantly altered in comparison with the control group (chi-square test , Yate's correction, p < 0.01).

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The inspection of the foetuses revealed one cleft palate in the 500 mg/kg dose group and 3 cleft palates (one litter affected) in the 1500 mg/kg dose group. From a series of untreated controls spontaneous cleft palate frequency was evaluated to be 0.25 % (5/2030 foetuses from 5 litters affected) vs. 1.2% (3/253 foetuses) in the high dose group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal assessment on foetuses of the three dose groups did not reveal any clearcut deviation from the vehicle control. One instance of irregularly-shaped sternum was observed in each the 500 mg/kg and the control group. Partial synostosis of ribs no. 7 + 8 was detected in one foetus of the 1500 mg/kg dose group.

Visceral malformations:

no effects observed

Description (incidence and severity):

The only pathological change of organ differentiation consisted of dilated renal pelvic cavitation in a foetus of the 500 mg/kg dose group.

Dose descriptor:

NOAEL

Effect level:

1 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981-05-12)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Cheshire Rabbit Farms (Dudden Lodge, Nr. Tarporley, Cheshire), Ranch Rabbits (Crawley Down, Sussex), Buxted Rabbit Co. Ltd. (Gt. Totease Fram, Buxted, Sussex)
- Weight at study initiation: 3.5 to 4.6 kg (pretest); 2.8 to 3.6 kg (females only, main study)
- Age at study initiation: 12 - 20 weeks
- Housing: individual housing in metal cages equipped with sheet stainless steel sides, stainless steel wire front and an aluminated perforated floor panel. Individual undercage plastic trays were lined with absorbent paper.
- Diet (e.g. ad libitum): SDS rabbit diet SQC, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose (1%)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The highest required concentration of suspension was prepared by trituration of weighed compound with vehicle using a mortar and pestle to form a smooth mixture, then addition of further vehicle to required volume. Further mixing was carried out using a "Silverson" Laboratory mixer. The lower concentrations were prepared by serial dilution with the vehicle.

VEHICLE
- Concentration in vehicle: 5 % (dose: 500 mg/kg bw/d), 10% (dose: 1000 mg/kg bw/d), 20% (dose: 2000 mg/kg bw/d)
- Amount of vehicle (if gavage): 10mL/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1
- Length of cohabitation: at least 1 hour after coitus was observed

Duration of treatment / exposure:

day 7-19 of gestation

Frequency of treatment:

daily, 7 days/week

Duration of test:

29 days after gestation

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

18 (female rabbits)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were based on a prior preliminary study in which animals were orally exposed to test substance with doses of 500, 1000 and 2000 mg/kg bw over a period of 13 days and sacrifice after 21 days. At 2000 mg/kg bw/d majority of animals showed pale discoloration of the faeces during the treatment period.

- Rationale for animal assignment (if not random):
rabbits were weighed and 24 within the bodyweight range 3.5 to 4.6 kg allocated to four groups, such that mean bodyweight and source of animals were similar for all groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, 7, 9, 11, 15, 20, 24 and 29 of gestation

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of pre-implantation loss: Yes
- Number of post-implantation loss: Yes
- Other:
- number and distribution of live young
- number and distribution of embryonic/foetal deaths
- individual foetal weights
- foetal abnormalities
- Embryonic/foetal deaths were classified as:
- Early: only placenta visible at termination
- Late: both placenta and embryonic remnants visible at termination
Abortion: only implantation site scars visible at termination

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 ]

OTHER:
Live young were examined externally then killed by intrathoracic injection of Pentobarbitone Sodium. They were weighed; dissected and examined for visceral abnormalities, and sexed. Where appropriate young showing suspected abnormalities were examined by alternative procedures (eg. micro-dissection, histopathology) to clarify initial observations. Young were skinned, eviscerated and fixed in 740P industrial methylated spirit; after fixation the heads were sliced through the line of the frontoparietal suture and the brain examined for visible abnormalities (eg.hydrocephaly, hydranencephaly) prior to clearing and staining (modified Dawson technique) of the carcasses for skeletal examination.

Statistics:

Statistical analyses were performed routinely on litter data and incidences of skeletal anomalies and skeletal variants using the litter as the basic sample unit. Non-parametric methods (Kruskal-Wallis and Jonckheere tests) were employed since these values rarely follow a normal distribution.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The majority of animals at 2000 mg/kg/day showed pale discolouration of faeces throughout the treatment period. At 1000 mg/kg/day approximately half of the animals occasionally showed pale discolouration of faeces during the treatment period. There were no other signs of reaction considered attributable to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Single dosed animals at 500 and 2000 mg/kg/day, and one in the control group died or were killed. No treatment relationship was considered to be indicated.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Intergroup variation in bodyweight gain generally appeared unrelated to dosage, although at all dosages mean gain during the first two days of treatment was slightly lower than among control animals. Overall weight gain at 1000 and at 2000 mg/kg/day was greater than that of control animals. At 1000 mg/kg/day weight gain was higher than in the high dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

At all dosages mean food consumption during the first four days of the dosing period was similar to that of control animals. Thereafter individual mean test group values tended to be higher than, or similar to, those of control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings at terminal autopsy that were considered attributable to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on maternal toxic effects:

Pregnancy rate as judged by the numbers of animals pregnant at termination, was high in all groups.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects at this dose level

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean foetal weights were essentially similar for all groups and intergroup differences- among mean values for litter weight were not statistically significant (P >0.05).

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Intergroup differences among mean values for litter size,and pre- and post implantation loss were not statistically significant (p >0.05).

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

A single animal, from the control group, showed total litter loss. For the following intergroup comparisons control values calculated excluding this animal (i.e. Mean B values) were employed.

External malformations:

no effects observed

Description (incidence and severity):

The incidence of malformed foetuses appeared unaffected by treatment; although the incidence at 500 mg/kg/day was higher than in the control group, the incidence at 1000 mg/kg/day was lower, and at 2000 mg/kg/day no malformations were observed.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Incidences of skeletal anomalies were not adversely affected at any dosage and the only differences to be statistically significantly (P <0.05) related to lower incidences of skeletal anomalies at 500 and 2000 mg/kg/day. At all dosages the incidence of foetuses with extra thoracolumbar rib and of foetuses with variant sternebrae tended to be similar to the control incidence; there were no statistically significant differences (p >0.05).

Visceral malformations:

no effects observed

Description (incidence and severity):

Incidences of visceral anomalies were not adversely affected at any dosage.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Abnormalities:

not specified

Developmental effects observed:

not specified