Registration Dossier

Administrative data

Description of key information

No repeated dose oral toxicity study with the test substance is available. An OECD 408 supported by an OECD 422 Guideline study in rats with 2,2’-azobis(isobutyronitrile) was used as a read across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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.
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
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
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Not available in English language
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

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
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.

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.
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)
Critical effects observed:
not specified
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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
23 JULY 2012 to 23 JANUARY 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current test guidelines and GLP compliant
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK
- Age at study initiation: 26 to 30 days old on arrival, approximately 5-7 weeks old at start of dosing
- Weight at study initiation: Males = 127.2 to 192.3 g, Females = 111.6 to 167.7 g at start of dosing
- Fasting period before study: No
- Housing: Animals housed in groups of two, in cages that conform with the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989). Bedding provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, UK)
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No 1, Expanded, (Special Diets Services Ltd, Witham, UK) ad libitum
- Water (e.g. ad libitum): Mains water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24C
- Humidity (%): 45 to 65%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: To: 04 September 2012 to 05 December 2012
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in corn oil following dispensary SOPs and the formulation method 8266542_O_01D. The Group 2 formulation for Week 3 of dosing was prepared differently to formulation method 8266542_O_01D. The test substance was weighed into a small beaker (due to no available large capacity beaker compatible with the balance), which was not large enough to take the vehicle as well. The test substance beaker was rinsed out thoroughly with the vehicle and the test substance and rinsings were added to the vehicle.
The formulations were stored refrigerated (2 to 8C) in a sealed container, protected from the light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil selected due to stability of test substance in the vehicle, and use in previous study R-95-007.
- Concentration in vehicle: 0.1 (low), 0.4 (intermediate) or 2.0 mg/mL (high)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKBH894V
- Purity: Not available - on file with the manufacturer
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HOMOGENEITY: Samples (three aliquots from the top and three from the bottom of the test substance formulations and two random aliquots from control/vehicle formulations) were taken in Week 1 and 13 for analysis of homogeneity.
CONCENTRATION VERIFICATION: Samples (three aliquots from the top and three from the bottom of test substance formulations and two random aliquots from control/vehicle formulations) were taken in Week 1 and 13 for analysis of achieved concentration. The mean of the homogeneity results was taken as the achieved concentration where the sampling occasion coincided.
Duration of treatment / exposure:
92 days
Frequency of treatment:
Animals dose once daily vial oral gavage
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
other: nominal in formulation
Remarks:
Doses / Concentrations:
2 mg/kg/day
Basis:
other: nominal in formulation
Remarks:
Doses / Concentrations:
0.5 mg/kg/day
Basis:
other: nominal in formulation
No. of animals per sex per dose:
10 male and 10 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: High dose (10 mg/kg/day) selected based on previously conducted study in this species (Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test of 2,2'-Azobis(2-methylpropionitrile) by Oral Administration in Rats; study number R-95-007).
Intermediate dose (2 mg/kg/day) was the approximate geometric mean between the low and high dose levels and was proposed in order to obtain a No Observed Adverse Effect Level (NOAEL) in the possible event that 10 mg/kg/day was not well tolerated after dosing. The low dose level (0.5 mg/kg/day) was proposed in order to try and obtain a NOEL.

- Rationale for animal assignment: Total randomisation procedure used to assign animals to treatment groups.
Positive control:
None used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Beginning and end of the working day for signs of ill health or overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights recorded on Day -7, on the first day of dosing, at weekly intervals and before necropsy.

FOOD CONSUMPTION:
The amount of food consumed by each cage of animals was determined weekly. Consumption was calculated as g/animal/day.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
Investigations performed on all animals pre-treatment and Group 1 and 4 animals in Week 12. A mydriatic agent was instilled into the eyes before examination.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the jugular vein of all animals in Week 13.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Haemoglobin, red blood cell (erythrocyte) count, haematocrit packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, haemoglobin ditribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were withdrawn from the jugular vein of all animals in Week 13.
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorous, creatinine, urea, glucose.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight from all animals in Week 12.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: Volume, colour, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals subjected to a battery of behavioural tests and observations in Week -1 and once weekly to the end of the dosing period.
- Dose groups that were examined: All animals
- Battery of functions tested:
Weekly observations - before removal from the cage: posture, activity, gait, tremor, convulsion, excessive vocalisation, arousal upon opening cage.
Weekly observations - after removal from the cage: Ease of removal, ease of handling, excessive vocalisation, tremor, convulsion, palpebral closure, exophthalmus, lacrimation, lacrimation type, salivation, respiration, piloerection, appearance of fur, other.
Open field observations (each animal was placed into an open field arena for 2 minutes): Latency to first step, posture, arousal, circling, gait type, gait type severity, stereotypy, tremor, convulsion, other.
Open field tests: Approach response, touch response, tail pinch, air righting ability, pupillary response, corneal tactile response, auditory startle response, hindlimb foot splay, forelimb and hindlimb grip strength.

OTHER:
Locomotor activity: Assessed for each anmal in an automated photocell activity recorder for 30 minutes on one occasion during Week 13. Activity counts were recorded at 2-minute intervals. The following parameters were determined: Total activity, total mobile counts.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
Immunhistochemical investigations: Additional sections of the kidney from all animals in Groups 1 to 4 were stained immunohistochemically for alpha-2-microglobulin. Immunohistochemical demonstration methods were used to demonstrate hyaline in the kidney. Histologically prepared sections of the kidney were immunostained using a commercial antibody to hyaline. Following incubation with the appropriate primary antibody, sections were then incubated with a suitable secondary biotinylated antibody and then visualised with a tertiary reagent and chromogen.
Statistics:
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3 and 4) were compared.
Body weight gains, food consumption, terminal body weights, haematology, clinical chemistry and urine analysis variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>0.05), and the group effect from the ANOVA was significant (P=0.05), pairwise comparisons with control were made using Dunnett's test.
Where Levene’s test was significant (P=0.05), a rank-transformation was applied to the data prior to analysis.
No analysis was performed for variables with values above or below the limit of the assay.
Some variables were not analysed due to there being too few values for meaningful analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA), for each sex separately, using the terminal body weight as the covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where there was no evidence of heterogeneity among the slopes (P>0.01), and the group effect from the ANCOVA was significant (P=0.05), pairwise comparisons with control were made using Dunnett's test.
Clinical signs:
no effects observed
Description (incidence and severity):
There were six decedents during the study. Animal 5M (control) died on Day 42, Animal 48F (control) died on Day 72, Animal 13M (given 0.5 mg/kg/day) died on Day 73, Animal 51F (given 0.5 mg/kg/day) died on Day 75, and Animal 1M (control) died on Day 84 of the study. In all these animals there were various inflammatory lesions in the thoracic cavity, including lung pleuritis, heart pericarditis and epicarditis, and serositis around the thymus and aorta. These findings were suggestive of death due to dosing errors and subsequent inflammatory changes, and were considered to be incidental deaths due to study procedures. For Animal 11M (given 0.5 mg/kg/day) the cause of demise was not determined. There were no deaths considered to be related to effects of AZDN. In addition to findings associated with terminal events, the range of macroscopic and microscopic findings in decedents was generally similar to those in animals surviving to terminal kill. For clinical signs, there were considered to be no effects of treatment. Clinical signs were observed but the type, severity and distribution were typical of rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
There were six decedents during the study. Animal 5M (control) died on Day 42, Animal 48F (control) died on Day 72, Animal 13M (given 0.5 mg/kg/day) died on Day 73, Animal 51F (given 0.5 mg/kg/day) died on Day 75, and Animal 1M (control) died on Day 84 of the study. In all these animals there were various inflammatory lesions in the thoracic cavity, including lung pleuritis, heart pericarditis and epicarditis, and serositis around the thymus and aorta. These findings were suggestive of death due to dosing errors and subsequent inflammatory changes, and were considered to be incidental deaths due to study procedures. For Animal 11M (given 0.5 mg/kg/day) the cause of demise was not determined. There were no deaths considered to be related to effects of AZDN. In addition to findings associated with terminal events, the range of macroscopic and microscopic findings in decedents was generally similar to those in animals surviving to terminal kill. For clinical signs, there were considered to be no effects of treatment. Clinical signs were observed but the type, severity and distribution were typical of rats of this age and strain.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males treated with 10 mg/kg/day showed a slight reduction in bodyweights at the end of the treatment period (-5.2% compared with controls) but differences were not statistically significant. There were no similar differences in females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Changes not considered to be of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Changes not considered to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Minor changes in urine parameters of males at all dose levels. Males at 10 mg/kg/day also had a paler colour urine and although there was a slight increase in volume, this was not statistically significant.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were considered to be no effects of treatment on any parameter of the Functional Obersvation Battery.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were treatment-related changes in liver and kidney weights when compared with concurrent controls, and these changes correlated with findings seen microscopically.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A higher level of pale and/or mottled kidneys recorded in all treated male groups. No other macroscopic findings considered related to treatment with AZDN. Findings related to treatment with AZDN were noted in the liver (both sexes) and in male kidneys.
Details on results:
ORGAN WEIGHTS:
Group mean adjusted liver weights were increased in all treated male groups (M: 8, 4 and 9%, respectively) and in females given 2 or 10 mg/kg/day (F: 9 and 23%, respectively). Equivalent liver to body weight ratios were also increased (M: 7, 2 and 10%, F: 8 and 24%, respectively) as were liver to brain weight ratios (M: 6, 4 and 4%, F: 9 and 20%, respectively) when compared with concurrent controls.
Group mean adjusted kidney weights were increased in all treated male groups (M: 9, 21 and 20%, respectively). Equivalent kidney to body weight ratios were also increased (M: 8, 19 and 20%, respectively) as were kidney to brain weight ratios (M: 7, 21 and 14%, respectively) when compared with concurrent controls. Kidney weights in treated females were similar to controls.
Thymus weights in treated males were generally decreased compared with concurrent controls, but in females thymus weights were increased compared with concurrent controls. As there was no microscopic correlation with these findings, and inconsistency between the sexes, these organ weight changes in the thymus were not considered to be related to treatment with AZDN.
All other organ weight and/or organ weight ratio changes, including those that were statistically significant, were attributed to normal biological variation and were considered not to be test-item related as they were either small in magnitude, not dose-dependent, inconsistent between the sexes, due to normal inter-animal variability and/or lacked a histopathological correlate.

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Macroscopic and Microscopic Observations

In the liver, hepatocyte hypertrophy was seen in all treated male groups and in females given 2 mg/kg/day (7 females grade 1, 1 female grade 2) or 10 mg/kg/day (10 females grade 2), and showed a dose-related trend. The finding correlated with the increase in liver weights noted at necropsy, and was characterised by an increase in size of hepatocytes, mainly in the centrilobular areas, with paler eosinophilic cytoplasm. In treated males there were also minor levels of hepatocyte vacuolation. This finding was characterised by generally large clear vacuoles in hepatocytes.

Incidence of hepatocyte hypertrophy and hepatocyte vacuolation: liver – terminal kill

 

 

Males

 

Females

 

 

1M

2M

3M

4M

 

1F

2F

3F

4F

Tissue and finding

Level (mg/kg/day)

0

0.5

2

10

 

0

0.5

2

10

 

 

 

 

 

 

 

 

 

 

 

Liver

No. examined

8

8

10

10

 

9

9

10

10

hepatocyte hypertrophy

Grade -

8

4

0

0

 

9

9

2

0

 

1

0

2

0

0

 

0

0

7

0

 

2

0

2

9

3

 

0

0

1

10

 

3

0

0

1

7

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

hepatocyte vacuolation

Grade -

8

7

1

5

 

9

9

10

10

 

1

0

1

9

5

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Key: “-“ =finding not present, 1 = minimal, 2 = slight, 3 = moderate.


In the kidney there were higher levels of hyaline droplets and tubular basophilia/focal nephropathy in all treated male groups compared with controls, and levels of the findings were similar in all three treated groups. These findings correlated with the increase in kidney weights and the pale and/or mottled kidneys noted at necropsy. Hyaline droplets were characterised by bright eosinophilic inclusions in the proximal tubular cells of the kidney. Tubular basophilia/focal nephropathy was characterised by groups of tubules with basophilic cytoplasm, degenerating and regenerating tubules, sometimes with dilated tubules, granular casts and inflammatory cell infiltrate.

Hyaline droplets were not seen in females and the levels of tubular basophilia/focal nephropathy in females given 10 mg/kg/day were similar to controls.

The immunohistochemical staining for alpha 2u globulin showed some background staining, possibly due to cross-reactivity of the antibody, but levels of staining above the background, localised in the proximal convoluted tubules, generally correlated with the levels of hyaline droplets seen in males. Therefore the hyaline droplets were considered to be due to accumulation of alpha 2u globulin.

Incidence of hyaline droplets and tubular basophilia/focal nephropathy: kidney – terminal kill

 

 

Males

 

Females

 

 

1M

2M

3M

4M

 

1F

2F

3F

4F

Tissue and finding

Level (mg/kg/day)

0

0.5

2

10

 

0

0.5

2

10

 

 

 

 

 

 

 

 

 

 

 

Kidney

No. examined

8

8

10

10

 

9

2

1

10

 hyaline droplets

Grade -

0

0

0

0

 

9

2

1

10

 

1

2

0

0

0

 

0

0

0

0

 

2

6

0

0

0

 

0

0

0

0

 

3

0

8

10

10

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

tubular basophilia/focal nephropathy

Grade -

6

0

0

0

 

8

1

1

9

1

2

0

1

1

 

1

1

0

1

 

2

0

5

3

6

 

0

0

0

0

 

3

0

3

6

3

 

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

Key: “-“ =finding not present, 1 = minimal, 2 = slight, 3 = moderate.

 

There were no other microscopic findings related to treatment with/administration of AZDN.

Microscopic findings in other tissues were generally infrequent, of a minor nature and consistent with the usual pattern of findings in rats of this strain and age.

Conclusions:
AZDN was well tolerated with minimal reaction to treatment and no evidence of neurotoxicity in either functional observation battery or locomotor activity. The NOAEL was considered to be 10 mg/kg/day.
Executive summary:

The objective of the study was to determine the toxicity of the test article, AZDN (2,2’-dimethyl-2,2’-azidopropionitrile), following daily oral (gavage) administration to the rat for 13 weeks.

The study was conducted at the request of the regulatory authorities in order to fulfill REACH requirements.

The oral route of administration was chosen because it is a likely human exposure route.

Groups ofratsof the Crl:CD(SD) strain were dosed as follows:

 

 

Number of Animals

Dose Level

Dose Concentration

Group

Subgroup

Male

Female

(mg/kg/day)

(mg/mL)

 

 

 

 

 

 

1 (Control)a

1 (Toxicity)

10

10

0

0

2 (Low)

1 (Toxicity)

10

10

0.5

0.1

3 (Intermediate)

1 (Toxicity)

10

10

2

0.4

4 (High)

1 (Toxicity)

10

10

10

2.0

 

 

 

 

 

 

a Group 1 received vehicle control article only

 

Assessment of toxicity was based on clinical signs, post dosing observations, functional observation battery and locomotor activity, body weights, food consumption, ophthalmoscopy, clinical pathology, organ weights and macroscopic and microscopic histopathology.

Incidental deaths due to study procedures were seen in controls and animals given 0.5 mg/kg/day, but there were no decedents during the study related to treatment with AZDN.

There were no clinical signs related to treatment and post dosing observations were consistent with reactions to taste of dosed materials. There were no toxicologically significant effects of treatment on ophthalmoscopy, functional observation battery, locomotor activity, bodyweights, food consumption or haematology.

There was a treatment related increase in creatinine in males. Females had higher calcium and lower chloride at all dose levels.

There were minor changes in urine parameters of males at all dose levels including reductions of urobilinogen and bilirubin scores, and increased amorphous debris scores. Males treated with 10 mg/kg/day also had paler colour urine and increased volumes.

Group mean adjusted liver weights were increased in all treated male groups and in females given 2 or 10 mg/kg/day. Group mean adjusted kidney weights were increased in all treated male groups. Kidney weights in treated females were similar to controls.

On macroscopic examination, a higher level of pale and/or mottled kidneys was recorded in all treated male groups.

On microscopic examination, hepatocyte hypertrophy was seen in the liver of all treated male groups and in females given 2 or 10 mg/kg/day, and showed a dose-related trend. There were also minor levels of hepatocyte vacuolation in the liver of treated males. In the kidney there were higher levels of hyaline droplets and tubular basophilia/focal nephropathy in all treated male groups compared with controls, and levels of the findings were similar in all three treated groups. Immunohistochemical staining confirmed the presence of alpha 2 microglobulin and therefore the hyaline droplets were considered to be due to accumulation of alpha 2u globulin.

In conclusion, daily administration of AZDN for 13 weeks at dose levels of 0.5, 2 or 10 mg/kg/day was well tolerated. There was no evidence of neurotoxicity. There were slight changes in some blood clinical chemistry and urine parameters at all dose levels. The liver and kidney were identified as target organs based on increased weights (at all dose levels in males) and microscopic findings of hepatocyte hypertrophy (at all dose levels in males and at =2 mg/kg/day in females) and focal nephropathy/tubular basophilia and hyaline droplets in males at all dose levels. The liver hypertrophy is considered an adaptive change commonly associated with metabolic burden and the changes in the kidney are considered specific to the male rat as confirmed by immunohistochemical staining. Therefore, the nature of these histopathological changes are considered to be of no toxicological relevance and the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 10 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The repeat dose toxicity of 2,2’-Azobis(2 -methylpropionitrile) was evaluated in a 90 days repeated dose toxicity study performed according to OECD 408 with the read-across substance. The NOAEL in the study was set at 10 mg/kg/day. No adverse effects relevant for Human risk assessment were reported.
The repeat dose toxicity of 2,2’-Azobis(2 -methylpropionitrile) was evaluated in a combined reproductive / developmental screening study following OECD 422 guideline with the read-across substance. NOEL for repeat toxicity was suggested to be less than 2 mg/kg/day in males and 2 mg/kg in females.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No repeated dose oral toxicity study with the test substance is available. An OECD 408 supported by an OECD 422 Guideline study in rats with 2,2’-azobis(isobutyronitrile) was used as a read across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances are similar in physicochemical properties, have common effects seen at acute exposures, and are similar in metabolic pathway.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

2,2’-Azobis(isobutyronitrile) was orally administered to male and female rats 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 order to evaluate the repeated dose toxicity and effects of the test item on the reproductive performance in parental animals and development and growth of the next-generation animals.Treatment with 2,2’-azobis(isobutyronitrile) inhibited weight gain and feed consumption in both males and females at 50 mg/kg.These changes were thought to be attributable to the treatment since similar changes were observed in the 50 mg/kg group in a preliminary 14-day repeated dose toxicity study.Weight gain was also inhibited at 10 mg/kg in females, suggesting the presence of gender differences.Observation of general signs and symptoms during the treatment period revealed scattered incidence of salivation immediately after administration in the 10 mg/kg and higher dose groups.Because a prompt recovery was noted in all cases, this change was not thought to be attributable any toxic potential effect.Necropsy revealed increases in weight of the liver in the 10 mg/kg and higher dose groups in males and in the 50 mg/kg group in females.Histopathological examinations showed centrilobular hypertrophy of hepatocytes in the 10 mg/kg and higher dose groups in both males and females.This change was thought to reflect increases in hepatic function induced by 2,2’-azobis(isobutyronitrile)dosing because it has been shown to be transferred to the liver after entering the body and therefore this change would represent an adaptative response of the liver. Blood biochemical examinations in the present study showed increases in total protein, albumin, and total cholesterol in males from the 50 mg/kg group.Increases in Ca concentrations observed in the 50 mg/kg group were thought to be associated with increases in albumin.Because the A/G ratio decreased in this group, blood globulin concentrations were also thought to have increased.In necropsy, weight of the kidneys showed a tendency to increase in males from the 2 mg/kg or higher dose groups and females from the 50 mg/kg group.Histopathological examinations revealed increases in eosinophilic bodies and regenerative renal tubules in males from the 2 mg/kg or greater dose groups.Both changes may spontaneously occur in males.However, their incidence and severity were apparently different from those seen in the control group.Granular casts containing necrotic cells were also observed.Coupled with the fact that inorganic P concentrations increased and Cl concentrations decreased in males from the 50 mg/kg group, these findings suggest that2,2’-azobis(isobutyronitrile)has effects on the kidneys, however considering the fact that only males were observed with renal changes, it is assumed that alpha-2-macroglobulin is involved in this specific toxicity. Increases in platelets observed in hematological examinations in males from the 50 mg/kg group were thought to be attributable to 2,2’-azobis(isobutyronitrile) because similar effects were seen in the 50 mg/kg group in the preliminary study, but the toxicological significance of this change could not be clarified in the present study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
NOAEL is based on OECD Guideline, GLP study in rats with read-across chemical, 2,2'azobis(isobutyronitrile)

Justification for classification or non-classification

Based on the lack of target organ effects in a repeated dose study with the read across chemical, 2,2'-azobis(isobutyronitrile), the test substance does not need to be classified for repeat dose toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.