Registration Dossier

Administrative data

Description of key information

Oral: OECD 401; rat LD50: 337 mg/kg. Reliability = 1
Dermal ( (based on read across to 2,2'-dimethyl-2,2'-azodipropiononitrile): OECD 402; rat LD50 >2000 mg/kg. Reliability = 1
Inhalation: similar to OECD 403; rat 4-hr LC50 >8.9 mg/L (8900 mg/m3). Reliability =2

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
Remarks:
The study was conducted according to guideline in effect at time of study conduct.
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
other: CD strain (remote Sprague-Dawley origin)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately five weeks
- Weight at study initiation: males from 110 - 144 g and for females from 105 - 130 g.
- Fasting period before study: yes
- Housing: The animals were housed in stainless steel grid cages measuring 54 x 33 x 20 cm. The grid floor ensured rapid removal of waste material to undertrays which were cleaned as necessary. Five animals of the same sex were accommodated in each cage, unless reduced by mortality. The cages were suspended in mobile stainless steel racks.
- Diet: A commercially-available complete pelleted rodent diet was fed without restriction, except for the removal of food approximately 18 hours before administration of the test material.
- Water: Animals had free access to tap water supplied in a single bottle per cage and re-filled as required.
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- All rooms were kept at slight positive pressure relative to the outside and each had its own filtered air supply giving approximately 15 complete air changes per hour without re-circulation. A temperature of 18 - 22°C and a relative humidity range of 32 - 48%. were achieved during the study. The relatively low humidity values did not overtly affect the health of the animals. Electric time-switches regulated a lighting cycle of 12 hours of artificial light per day.
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose
Details on oral exposure:
VEHICLE
- Concentration in vehicle: The test material was prepared at appropriate concentrations in 0.5% w/v methylcellulose in purified water (obtained through the reverse osmosis of tap water).


MAXIMUM DOSE VOLUME APPLIED: volume-dosage of 20 ml/kg

Doses:
202, 254, 320 and 402 mg/kg bodyweight
No. of animals per sex per dose:
5/sex/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: Three separate inspections were made during the first hour after dosing and two further inspections during the remainder of Day 1. From Day 2 onwards, the animals were inspected twice daily (morning and afternoon). The type, time of onset and duration of reactions to treatment and the circumstances of any death were recorded
-Frequency of weighing: The bodyweight of each animal was recorded on the day before dosing and on Days 1, 8 and 15. The test was terminated on the morning of Day 15.
- Necropsy of survivors performed: yes
Statistics:
Probit analysis by the method of Finney was used to determine the acute median lethal dosage, 95% confidence interval and slope of the dose response curve of the test material for both sexes. The calculations were performed by the GLIM statistics program using a special macro program developed by Baker.
Sex:
male
Dose descriptor:
LD50
Effect level:
334 mg/kg bw
Based on:
test mat.
95% CL:
278 - 390
Sex:
female
Dose descriptor:
LD50
Effect level:
333 mg/kg bw
Based on:
test mat.
95% CL:
271 - 395
Sex:
male/female
Dose descriptor:
LD50
Effect level:
337 mg/kg bw
Based on:
test mat.
95% CL:
306 - 368
Mortality:
No animals died at 202 or 254 mg/kg.
Four males and 1 female died at 320 mg/kg.
Three males and 5 females died at 402 mg/kg
Clinical signs:
Ante mortem signs comprised lethargy, reduced mobility, staggering gait, reddening, muscle tremor, piloerection, salivation and hunched posture.

Signs of reaction in surviving animals were similar to those of the decedents with the addition of irritability, breathing irregularities, ungroomed appearance, diarrhoea, pigmented orbital secretion, pigmented staining of the snout and closed eyes. All the surviving animals were overtly normal by the sixth day.
Body weight:
The surviving animals acheived expected bodyweight gains.
Gross pathology:
Necropsy of the decedents revealed localised yellow, red or brown fur staining, abnormal gastro-intestinal contents and a single observation of dark areas on the glandular mucosa of the stomach. Necropsy of the surviving animals recorded no significant macroscopic lesions.

Mortality data

 

 

Day

Dose mg/kg

Sex

 Mortality: Dead/Alive

 

 

1

2

3

4

14

202

M

0/5

0/5

0/5

0/5

0/5

F

0/5

0/5

0/5

0/5

0/5

254

M

0/5

0/5

0/5

0/5

0/5

F

0/5

0/5

0/5

0/5

0/5

320

M

0/5

4/5

4/5

4/5

4/5

F

0/5

1/5

1/5

1/5

1/5

402

M

0/5

3/5

3/5

3/5

3/5

F

1/5

5/5

5/5

5/5

5/5

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Combined sex LD50 = 337 mg/kg.
Executive summary:

The acute oral toxicity of the test substance was investigated in four groups of five male and five female CD rats. The test material was administered at dosages in the range 202 - 402 mg/kg, at a volume-dosage of 20 mL/kg in 0.5% w/v aqueous methylcellulose. Mortality and signs of reaction to treatment were recorded during a subsequent 14-day observation period. The surviving animals were killed on the following day. All animals were subjected to necropsy. No deaths ocurred at the 202 and 254 mg/kg dose. Mortalities were noted at the two higher doses of 320 and 402 mg/kg. All deaths ocurred on the day of dosing or during the first overnight period. The combined LD50 was determined to be 337 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
337 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: low respirability of atmosphere noted. 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.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
yes
Remarks:
only males were exposed
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 week
- Weight at study initiation: 232-250 g
- Fasting period before study: No
- Housing: Singly in stainless steel, wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not reported
- Humidity (%): Not reported
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Not reported
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: air
Details on inhalation exposure:
Generation: Dust atmospheres were generated with a 3-stage, hourglass-shaped system. A round flask at the bottom of the generator served as a dust reservoir. A cyclone-shaped flask, inserted above the reservoir, served as an elutriator. A cylindrical flask, inserted between the elutriator and the exposure chamber, served as a settling chamber to remove large particles. A motorized stirring rod with plastic paddles agitated dust in the generator. During the last exposure, surface vibrators were added to keep dust from accumulating on the generator’s walls. Air introduced as the bottom of the generator carried dust particles upward to the elutriator. Dilution air introduced at the elutriator swept airborne dust through the settling chamber and into the exposure chamber. Dust concentration was controlled by varying the two airflows.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
At 15- to 30-minute intervals, calibrated volumes of test atmosphere were drawn through pre-weighed glass fiber filters. Filters were weighed on a Cahn 26 Automatic Electrobalance®. Atmospheric concentration was determined from filter weight gain.
Duration of exposure:
4 h
Concentrations:
1.8, 3.7, and 8.9 mg/L
No. of animals per sex per dose:
6 males per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days, weekends included when deemed necessary
- Frequency of observations and weighing: Daily
- Necropsy of survivors performed: No
Statistics:
None reported
Sex:
male
Dose descriptor:
other: ALC
Effect level:
> 8.9 mg/L air
Exp. duration:
4 h
Mortality:
No mortality was observed.
Clinical signs:
During exposure, rats’ faces were covered with test material. Rats exposed to 1.8 mg/L exhibited no response to sound. At higher concentrations, observations could not be made due to the dense dust atmosphere in the chamber. Rats exposed to 1.8 and 3.7 mg/L exhibited red to brown ocular and/or nasal discharge for 1 day post exposure. No other adverse clinical signs were observed.
Body weight:
All rats exhibited slight to severe weight loss 1 day post exposure. At 8.9 mg/L, 1 rat continued to lose weight for 1 more day. Weight loss was followed by normal weight gain.
Gross pathology:
Not performed.
Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The lethal concentration of the test substance is greater than 8.9 mg/L.
Executive summary:

Groups of 6 male Crl:CD® rats were exposed to dust atmospheres of the test substance for single, 4-hour periods. The lethal concentration of the test substance as supplied is greater than 8.9 mg/L, the highest concentration that could be generated. Because of the atmospheres' low respirability, this test should be considered a hazard evaluation of the material as supplied, rather than an accurate toxicity evaluation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
8 900 mg/m³

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
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 is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction). 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. Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 8 weeks old
- Weight at study initiation: 371 ± 9 g for the males and 235 ± 7 g for the females
- Fasting period before study: none
- Housing: polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm)
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): drinking water filtered by a FG Millipore membrane (0.22 micron)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: From 03 March 2010 to 17 March 2010
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: 5 cm x 7 cm for the males and 5 cm x 6 cm for the females
- % coverage: 10% of the total body surface of the animals
- Type of wrap if used: gauze pad held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing and a restraining bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): any residual test item was removed using a dry cotton pad
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg
- For solids, paste formed: yes (substance moistened with 2 mL of purified water)

Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
other: historical control animals
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: the animals were observed frequently during the hours following administration of the test item, and then at least once a day until day 15. Animals were weighed individually just before administration of the test item on day 1 and then on days 8 and 15.
- Necropsy of survivors performed: yes
Statistics:
no
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
None.
Clinical signs:
None.
Body weight:
When compared to historical control data, a lower body weight gain was noted in all the males (16 to 35 g vs. 47 g ± 7 g in historical data base) and 1/5 females (7 g vs. 25 g ± 11 g in historical data base) between day 1 and day 8; returning to normal thereafter.
Gross pathology:
Macroscopic examination of the main organs of the animals revealed no apparent abnormalities.
Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Under the experimental conditions of this study, the dermal LD50 of the test item AZDN (batch No. 6089, purity: 99.2%) was higher than 2000 mg/kg in rats.
Executive summary:

The acute dermal toxicity of the test item, AZDN, was evaluated in rats according to OECD (No. 402, 24th February 1987) and Commission Regulation (EC) (No. 440/2008, Part B.3, 30 May 2008) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

The test item was applied to the skin of one group of ten Sprague-Dawley rats (five males and five females). The application was performed with the test item in its original form at the dose-level of 2000 mg/kg. The test site was then covered by a semi-occlusive dressing for 24 hours. Clinical signs, mortality and body weight gain were checked for a period of 14 days following the single application of the test item.

All animals were subjected to necropsy.

 

Results

No mortality, no clinical signs and no cutaneous reactions were observed during the study. When compared to historical control data, a lower body weight gain was noted in all the males (16 to 35 g vs.47 ± 7 g in historical data base) and 1/5 females (7 g vs. 25 g ± 11 g in historical data base) between day 1 and day 8; returning to normal thereafter. No apparent abnormalities were observed at necropsy in any animal.

 

Conclusion

Under the experimental conditions of this study, the dermal LD50 of the test item AZDN (batch No. 6089, purity: 99.2%) was higher than 2000 mg/kg in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw

Additional information

The rat oral LD50 was 337 mg/kg.The 4-hour inhalation LC50 was >8.9 mg/L (8900 mg/m3). No data were available for acute dermal toxicity with the test substance, but a dermal LD50 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) isthatthe substances are similar based on evidence from models to estimate dermal absorption. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach. Therefore, it is reasonable to read-across the information from the acute dermal study on 2,2’-azobis(isobutyronitrile) to address the data gap for the test substance. The rat dermal LD50 for 2,2’-azobis(isobutyronitrile) is >2000 mg/kg.


Justification for selection of acute toxicity – oral endpoint
OECD guideline, GLP study

Justification for selection of acute toxicity – inhalation endpoint
Scientifically valid study, similar to OECD guideline

Justification for selection of acute toxicity – dermal endpoint
LD50 is based on read across to acute inhalation study with 2,2'-dimethyl-2,2'-azodipropiononitrile (OECD guideline, GLP study).

Justification for classification or non-classification

Based on the acute oral LD50 in rats of 337 mg/kg, the test substance is classified as Xn, R22 (harmful if swallowed) for acute toxicity according to EU Directive 67/548/EEC and Cat 4 (H302: harmful if swallowed) for acute oral toxicity according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) regulation (EC) No. 1272/2008.

Based on the inhalation 4-hour LC50 in rats of >8.9 mg/L (8900 mg/m3) and the dermal LD50 in rats with 2,2’-azobis(isobutyronitrile) of >2000 mg/kg, no classification is required for acute inhalation or dermal endpoints according to the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.