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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
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 oral exposure:
Not available in English language
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

Doses / concentrations
Remarks:
Doses / Concentrations:
2, 10, 50 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
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. Additionnally for females, days 0 & 4 of lactation.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All males (13 per dose group)
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: All males (13 per dose group)
- Parameters checked in table 1 were examined.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following tissues were prepared for microscopic examination :
Heart, thymus, spleen AND liver, kidney, adrenal gland, testis, epididymis. These were also weighed.

Other examinations:
See the chapter 7.8.1 for the exams performed concerning reproductive toxicology.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Males : Temporary salivation after each administration was observed in the 10 mg/kg or more groups.
Females : One female died on post-partum day 3 in the 50 mg/kg group.

BODY WEIGHT AND WEIGHT GAIN
Males : Suppression of body weight gain during the early administration period was noted in the 50 mg/kg group.
Females : Slight decreases in body weight gain and food consumption during the early administration period were observed in the 10 mg/kg or moregroups. In addition, body weight gain and food consumption were decreased during pregnancy in the 50 mg/kg group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males : Suppression of food consumption during the early administration period was noted in the 50 mg/kg group.
Females : No effects.

HAEMATOLOGY
Males : Elevated platelet and white blood cell counts were apparent after the administration of the compound at the dose level of 50 mg/kg.
Females : No effects.

CLINICAL CHEMISTRY
Males : Increases in total protein and concentrations of albumin, total cholesterol, Ca and inorganic phosphorus and decreases in the A/G ratio an Cl concentration were apparent after the administration of the compound at the dose level of 50 mg/kg.
Females : No effects.

ORGAN WEIGHTS
Males : Increases in the kidney weights in all the treated groups and in the liver weights in the 10 mg/kg or more groups were observed at autopsy.
Females : Liver and kidney weights showed a tendency for increase in the 50 mg/kg group (observed at autopsy on post-partum day 4).

GROSS PATHOLOGY
No effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
Males : Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of males treated with the compound atany dose levels tested were noted, as well as granular casts in the lower nephrons. Centrilobular hypertrophy of hepatocytes was also detected in the 10 mg/kg or more groups.
Females : Centrilobular hypertrophy of hepatocytes was observed in the 10 mg/kg or more groups.

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 both sexes, based on liver toxicity.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
< 2 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Histopathology (kidneys)
Dose descriptor:
NOEL
Effect level:
2 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Body weight; food consumption; histopathology (liver)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The NOEL for toxicity was suggested to be less than 2 mg/kg/day in males and 2 mg/kg in females by the authors.
Considering the fact that only males were observed with renal changes, it is assumed that alpha-2-macroglobulin is involved in the specific toxicity observed. Then, the NOEL for the test item is 2 mg/kg/day for both sexes, based on liver toxicity.
Executive summary:

A study was conducted at Hatano Research Institute, Japan to evaluate the repeated dose toxicity and effects of the test substance 2,2’-Azobis(2-methylpropionitrile) (ABMPN) on the reproductive performance in parental animals and development and growth of F1 pups until day 4 of lactation. The study was conducted according to GLP and to OECD Guidelines for Testing of Chemicals: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (March 22, 1990).

The study design consisted of 13 male and 13 female Sprague-Dawley (Crj:CD) rats per group, dosed at 0 (vehicle control), 2, 10 and 50 mg/kg for 2 weeks before mating and for 2 weeks after mating, followed by additional 2 weeks after completion of mating in males and throughout the gestation period until Day 3 of lactation after parturition in females. 

In the males, ABMPN induced transient salivation at 10 mg/kg or greater doses and inhibited weight gain and feed consumption at an early stage of the treatment at 50 mg/kg. Necropsy conducted after repeated administration of 42 doses revealed increases in kidney weight at 2 mg/kg or greater doses and increases in liver weight at 10 mg/kg or greater doses. Findings in histopathological examinations included increases in eosinophilic bodies and basophilic renal tubules and the presence of granular casts at 2 mg/kg or greater doses, as well as centrilobular hypertrophy of hepatocytes at 10 mg/kg or greater doses. Hematological examinations of blood samples taken during necropsy showed increases in platelets and WBC at 50 mg/kg. Blood biochemistry revealed increases in the concentrations of total protein, albumin, total cholesterol, calcium and inorganic phosphorus, as well as decreases in the A/G ratio and Cl at 50 mg/kg.

In the females, ABMPN inhibited weight gain and feed consumption at an early stage of treatment at 10 mg/kg or greater doses. At 50 mg/kg, it also inhibited weight gain and feed consumption during gestation. One animal died 3 days after parturition. Necropsy conducted 4 days after parturition revealed a tendency toward increases in weight of the liver and kidneys. Findings obtained in histopathological examinations suggested centrilobular hypertrophy of hepatocytes at 10 mg/kg or greater doses.

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

The findings in offspring showed that ABMPN had no effects on the parturition index, live pup delivery index, overall delivery index, or the sex ratio and body weight on Day 0. 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 ABMPN groups showed any morphological anomaly.

It was concluded that under the conditions of the present study, the NOEL for parental toxicity of ABMPN is slightly lower than 2 mg/kg/day in males and 2 mg/kg/day in females and that the NOEL for reproduction/developmental toxicity is 50 mg/kg/day in males and 10 mg/kg/day in females and offspring.