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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP guideline study with acceptable restrictions. Restrictions : no english data on study design, no details on functional observational battery tests.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2'-AZOBIS(2-METHYLPROPIONITRILE)
- Physical state: white crystal
- Analytical purity: 99.9%
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Not available in English language.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
No english data

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males were exposed for 42 days.
Females were exposed from 14 days prior to mating to day 3 of lactation.
Terminal killing at day 43 for males and on day 4 of lactation for females.

Frequency of treatment:
No English data
Details on study schedule:
No English data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2 mg/kg bw (low dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg bw (intermediate dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw (high dose)
Basis:
actual ingested
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week the first week ; weekly afterwards, including during the pregnancy of females. Additionally for females, days 0 & 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption determined in four day blocks during the first week and weekly afterwards.
- Compound intake calculated in mean diet as g food/rat/average over the period of calcul.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality (at day 4), body weight recorded at day 0 and day 4.


GROSS EXAMINATION OF DEAD PUPS:
yes, morphological (no more data - this information comes from the result in the publication indicating "no morphological abnormalities")
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at day 43.
- Maternal animals: All surviving animals day 4 post partum.


GROSS NECROPSY
no data.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination :
Heart, thymus, spleen
AND Liver, kidney, adrenal gland, testis, epididymis. These ones were also weighed.
Postmortem examinations (offspring):
SACRIFICE
- Supposed at day 4. Not accurately indicated.

GROSS NECROPSY
- Gross necropsy consisted of external examinations as supposed by the result in the publication indicating "no morphological abnormalities".

HISTOPATHOLOGY / ORGAN WEIGHTS
No
Statistics:
No data
Reproductive indices:
Copulation index, Fertility index, Gestation index, Duration of pregnancy.

Offspring viability indices:
Viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Males: Temporary salivation after each administration was observed in the animals exposed at 10 mg/kg and more.
Females: One female in the 50 mg/kg group died on post-partum day 3.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: Suppression of body weight gain and food consumption during the early administration period were noted in the 50 mg/kg group.
Females: Slight decrease in body weight gain and food consumption during the early administration period were observed in the animals exposed at 10 mg/kg and more.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The compound showed no adverse effects on copulation and fertility, duration of pregnancy, gestation index and parturition at any of the dose levels tested. Three dams in the 50 mg/kg group showed abnormal lactation. (In the Japan HPV document, it was more accurately reported that 3 dams of 12 showed difficulty of nursling and two of them let all their offsprings die within the first 4 days after birth).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males: At autopsy, increases in the kidney weights in all of the compound-treated groups and in the liver weights in the animals exposed at 10 mg/kg and more were observed.
Females: At autopsy on post-partum day 4, liver and kidney showed a tendency for increase after the administration of the compound at the dose level of 50 mg/kg.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males: Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of all the animals exposed were noted, as well as granular casts in the lower nephrons. Centrilobular hypertrophy of hepatocytes was also detected in the groups of animals exposed at 10 mg/kg and more.
Females: Centrilobular hypertrophy of hepatocytes was observed in the groups of animals exposed at 10 mg/kg and more.

The NOEL for toxicity was suggested by the authors to be less than 2 mg/kg/day in males and 2 mg/kg in females. In the Japan HPV document, as renal pathological changes were observed only in males, accumulation of alpha-2-macroglobulin was suspected as a cause of male specific renal toxicity. Therefore, based on pathological changes in liver of both sexes, NOEL was considered to be 2 mg/kg/day for rats of both sexes, based on liver toxicity.

For reproductive and developemental toxicity the NOEL was 50 mg/kg/day in males and 10 mg/kg in females

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
2 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
2 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: General Toxicity body weight; food consumption ; histopathology (centrilobular hypertrophy of hepatocytes).
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No effect on reproductive toxicity in male
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: At 50 mg/kg bw/d, abnormal lactation in 3 dams

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

The compound did not demonstrate any adverse effects on viability (statistically), sex ratio and body weight gain of pups.
However, viability of newborns at birth and body weight of nurslings on postnatal day 4 in the 50mg/kg bw group were lower than controls.

For reproductive and developemental toxicity the NOEL was 10 mg/kg in pups according to the authors.

In Japan HPV document, it was considered that the effects on pups were caused by maternal toxicity since a difficulty in nursling (lactation) was reported. Then the NOAEL for reproduction in offspring was considered to be 50 mg/kg.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
10 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: decreased viability index and body weight on day 4 at 50 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Summary of developement of pups

Dose (mg/kg)

0

2

10

50

Number of pregnant females

12

12

11

12

Number of pregnant females with pup alive

12

12

11

12

Gestation Index

100

100

100

100

Gestation length in days

22.3 +/-0.5 (12)

22.1 +/-0.3 (12)

22.2 +/-0.4 (11)

22.4 +/-0.5 (12)

Number of corpora lutea

18.8 +/-2.5 (12)

17.8 +/-1.5 (12)

18.1 +/-2.0 (11)

16.9 +/-2.0 (12)

Number of implantation sites

17.3 +/-25 (12)

16.8 +/-1.5 (12)

17.1 +/-2.1 (11)

15.6 +/-1.4 (12)

Implantation index

92.9 +/-10.6 (12)

95.0 +/-5.7 (12)

94.7 +/-7.7 (11)

92.8 +/-9.3 (12)

Day 0 of lactation

 

 

 

 

Number of pups born

15.4 +/-2.5 (12)

15.8 +/-1.9 (12)

15.5 +/- 1.8 (11)

14.8 +/-1.5 (12)

Delivery index

88.8 +/-6.2 (12)

94.0 +/-6.1 (12)

91.6 +/-10.3 (11)

94.6 +/-4.4 (12)

Number of pups alive

15.0 +/-2.5 (12)

15.8 +/-1.9 (12)

15.5 +/-1.8 (11)

14.8 +/-1.5 (12)

Birth index

86.6 +/- 8.3 (12)

93.5 +/- 5.8 (12)

91.6 +/-10.3 (11)

94.6 +/-4.4 (12)

Live birth index

97.4 +/-4.9 (12)

99.4 +/- 1.9 (12)

 100.0 +/-0.0 (11)

100.0 +/- 0.0 (12)

Pup weight in grams

 

 

 

 

Male

6.2 +/-0.6 (12)

6.3 +/-0.5 (12)

6.3 +/-0.4 (11)

6.1 +/-0.2 (12)

Female

5.9 +/-0.5 (12)

6.0 +/-0.5 (12)

6.0 +/-0.3 (11)

5.9 +/-0.2 (12)

Sex ratio

50.5 +/-9.9 (12)

43.9 +/-12.8(12)

54.4 +/-10.9 (11)

53.6 +/-11.7 (12)

Day 4 of lactation

 

 

 

 

Number of pups alive

14.6 +/-2.3 (12)

15.6 +/-1.9 (12)

15.5 +/-1.8 (11)

11.6 +/-5.9 (12)

Viability index

 97.5 +/-4.1 (12)

99.0 +/-2.4 (12)

100.0 +/-0.0 (11)

77.9 +/-38 (12)

Pup weight in grams

 

 

 

 

Male

9.8 +/-1.4 (12)

9.8 +/-1.1 (12)

9.8 +/-1.0 (11)

9.0 +/-1.2 (12)

Female

9.5 +/-1.2 (12)

9.5 +/-1.1 (12)

9.4 +/-1.0 (11)

8.5 +/-1.1 (12)

Gestation Index: Number of pregnant female with pups alive/Number of pregnant females

Implantation Index: Number of implantation sites/Number of corpora lutea *100 (%)

Delivery Index: Number of pups born/Number of implantation sites *100 (%)

Birth Index: Number of pups alive on day 0/ Number of implantation sites *100 (%)

Live Birth Index: Number of pups alive on day 0/ Number of pups born *100 (%)

Sex ratio: Number of male pups alive on day 0/ Number of pups alive on day 0 *100 (%)

Viability index: Number of pups alive on day 4/ Number of pups alive on day 0 *100 (%)

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
In conclusion, the NOEL for reproductive and developemental toxicity was 50 mg/kg/day in males and 10 mg/kg in females and in pups.
According to CLP criteria, the test substance is not classified.
Executive summary:

The reproductive / developmental toxicity of 2,2'-Azobis(2-methylpropionitrile) was evaluated in male and female rats after oral administration (gavage) at doses of 0, 2, 10 and 50 mg/kg/day until day 43 for males and day 4 of post partum for females.

AZDN had no effects on the copulation index or fertility index at 50 mg/kg or lower doses. AZDN did not affect the length of the gestation period or delivery index in maternal animals, either. No abnormal parturition was observed. Abnormal nursing behaviour was noted in 3 animals from the 50 mg/kg group.

The findings in offspring showed no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0 at all dose-levels. In the 50 mg/kg group, the offspring viability index and body weight on Day 4 of lactation showed a tendency to decrease. No offspring from the AZDN groups showed any morphological anomaly.

Based on these results, the NOEL for reproductive and developmental toxicity was 50 mg/kg/day in males and 10 mg/kg in females and in pups.