2,2'-dimethyl-2,2'-azodipropiononitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

other information

Study period:

1997

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Data source

Reference

Reference Type:

secondary source

Title:

no data

Author:

MHW, Japan

Year:

1997

Bibliographic source:

cited in OECD SIDS Final 08/02

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

- males: 42 days
- females: from 14 days before mating to day 3 of lactation

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

13

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

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 and 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.

HAEMATOLOGY: Yes
- How many animals: all males (13 per dose group)

CLINICAL CHEMISTRY: Yes
- How many animals: all males (13 per dose group)

Sacrifice and pathology:

Terminal killing at day 43 for males and on day 4 of lactation for females.

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: Temporary salivation was induced at 10 mg/kg or more groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Females: One animal died on postpartum day 3 at 50 mg/kg.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Decrease in body weight gain was observed at 50 mg/kg.
Females: Decrease in body weight gain was observed in 10 mg/kg and more groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: Decrease in food consumption was observed in 15 mg/kg.
Females: Decrease in food consumption was observed in 10 mg/kg and more groups.

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

Description (incidence and severity):

Males: In blood analysis, there were several changes in 50 mg/kg group, such as an elevation of platelet and white blood cell counts, increases in total protein, albumin, total cholesterol, Ca and inorganic phosphorus, and decreases in the A/G ratio and Cl concentration.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Increases in eosinophilic bodies and basophilic changes of the renal tubular epithelial cells were observed in all treatment groups and granular casts in the lower nephrons were observed in 10 mg/kg and more groups. As these pathological changes were observed only in males, accumulation of α2u-macroglobulin is suspected as a cause of male specific renal toxicity. Centrilobular hypertrophy of hepatocyte was observed in 10 and 50 mg/kg groups (±: 4 in 13, +: 9 in 13 for 10 mg/kg, ++: 13 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).
Females: Centrilobular hypertrophy of hepatocytes was observed in 10 and 50 mg/kg groups (±: 6 in 13, +: 1 in 13 for 10 mg/kg, ±: 1 in 13, +: 11 in 13, ++: 1 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: In kidneys, absolute and relative weight was increased in all treatment group and in 10 mg/kg or more groups, respectively. Liver weights significantly increased by 14 and 66 % for absolute weight (14 and 74 % for relative weight) in 10 and 50 mg/kg group, respectively.
Females: In kidneys, absolute and relative weights were increased at 50 mg/kg. Liver weights significantly increased by 43 % for absolute weight (51 % for relative weight) in only 50 mg/kg group.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males: Increased eosinophilic bodies and basophilic changes of the renal tubular epithelial cells in the kidneys of males treated with the compound at any dose level 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.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day (nominal)

Sex:

male/female

2

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Males:

Temporary salivation was induced at 10 mg/kg or more groups. Decrease in body weight gain and food consumption was observed at 50 mg/kg. In kidneys, absolute and relative weight was increased in all treatment group and in 10 mg/kg or more groups, respectively. In addition, increases in eosinophilic bodies and basophilic changes of the renal tubular epithelial cells were observed in all treatment groups and granular casts in the lower nephrons were observed in 10 mg/kg and more groups. As these pathological changes were observed only in males, accumulation of α2u-macroglobulin is suspected as a cause of male specific renal toxicity. Liver weights significantly increased by 14 and 66 % for absolute weight (14 and 74 % for relative weight) in 10 and 50 mg/kg group, respectively. Centrilobular hypertrophy of hepatocyte was observed in 10 and 50 mg/kg groups (±: 4 in 13, +: 9 in 13 for 10 mg/kg, ++: 13 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups). In blood analysis, there were several changes in 50 mg/kg group, such as an elevation of platelet and white blood cell counts, increases in total protein, albumin, total cholesterol, Ca and inorganic phosphorus, and decreases in the A/G ratio and Cl concentration.

Females:

One animal died on postpartum day 3 at 50 mg/kg. Decrease in body weight gain and food consumption was observed in 10 mg/kg and more groups. In kidneys, absolute and relative weights were increased at 50 mg/kg. Liver weights significantly increased by 43 % for absolute weight (51 % for relative weight) in only 50 mg/kg group. However, centrilobular hypertrophy of hepatocytes was observed in 10 and 50 mg/kg groups (±: 6 in 13, +: 1 in 13 for 10 mg/kg, ±: 1 in 13, +: 11 in 13, ++: 1 in 13 for 50 mg/kg, compared to no changes in 0 and 2 mg/kg groups).

Applicant's summary and conclusion

Conclusions:

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 to evaluate the repeated dose toxicity and effects of the test substance 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, the test substance 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 cholestrol, calcium and inorganic phosphorus, as well as decreases in the A/G ratio and CI at 50 mg/kg.

In the females, the test substance 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 reveales 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 the test substance had no effects on the copulation index or fertility index at 50 mg/kg or lower doses. The test substance did not affect the length of the gestation period or delivery index in the 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 the test substance had no effects on the parturition index, live pup 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 test substance groups showed any morphological anomaly.

It was concluded that under the conditions of the present study, the NOEL for parental toxicity of the test substance 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.