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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 July 1996 - 05 August 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study (OECD 406)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study performed before the implementation of the REACH regulation.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2'-AZOBIS(ISOBUTYRONITRILE)
- Physical state: white powder
- Analytical purity: 99.2%
- Purity test date: 03/27/95
- Lot/batch No.: 721
- Expiration date of the lot/batch: 04/1997
- Storage condition of test material: in dark and at temperature of +4°C

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Lebeau, 78950 Gambais, France.
- age at study initiation: 3 months
- Weight at study initiation: 324 +/- 23 g for males and 342 +/- 20 g for females
- Housing: individually in suspended wire-mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days before the beginning of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
- Administration by intradermal route: The concentration chosen was 0.1% (w/w).
- Application by cutaneous route: The concentration chosen for the topical application of the induction phase and for the challenge application was
100%.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
- Administration by intradermal route: The concentration chosen was 0.1% (w/w).
- Application by cutaneous route: The concentration chosen for the topical application of the induction phase and for the challenge application was
100%.
No. of animals per dose:
- Control group: 5 males and 5 females.
- Treated group: 10 males and 10 females.


Details on study design:
RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study:
- By intradermal route, the maximal concentration which could pass through a needle was 0.1% (w/w). This concentration was well-tolerated and
induced 24, 48 hours and 6 days after the injection only a slight irritation.
- By cutaneous route, the undiluted test substance was applied for 24 hours under occlusive dressing to both flanks of one male and one female rabbit. No cutaneous reactions were observed 24 and 48 hours after removal of the dressing indicating that the concentration of 100% was
well-tolerated.
MAIN STUDY
A. INDUCTION EXPOSURE

Intradermal injections:
On Day 1, the treated group received three pairs of intradermal injections of 0.1 ml in the shoulder region which was cleared of hair so that one of each pair lies on each side of the midline.
Injection 1 was a 50:50 mixture (v/v) FCA/ physiological saline
Injection 2 was the test substance at 0.1% (w/w) in paraffin oil
Injection 3 was a 50:50 mixture (w/v) of 1 and 2
On Day 1, the control group received three pairs of intradermal injections of 0.1 ml in the same sites as in the treated animals.
Injection 1 was a 50:50 mixture (v/v) FCA/ physiological saline
Injection 2 was paraffin oil
Injection 3 was a 50:50 mixture (w/v) of 1 and 2

Cutaneous Applications:
On Day 7, the test areas of the treated and control groups were clipped. As the substance was not a skin irritant, the animals were treated with 0.5 ml of 10% sodium lauryl sulfate in vaseline, in order to create a local irritation.
On Day 8, the treated group received 500 mg of the undiluted test substance which was held in contact with the test area by an occlusive dressing for 48 hours.
On day 8, the control group received 0.5 ml of paraffin oil in a similar manner.


B. CHALLENGE EXPOSURE

On day 22, the animals from both groups received an application of 500 mg of the test substance undiluted to the posterior right flank and 0.5 ml of the vehicle to the posterior left flank. The patches were held in contact by an occlusive dressing for 24 hours.

24 hours after removing the patch, the skin reaction was observed and recorded according to the Magnusson and Kligman grading scale (see below). Approximately 24 hours after this observation a second observation (72 hours) was made and once again recorded.
Challenge controls:
no data
Positive control substance(s):
yes
Remarks:
2,4-dinitro chlorobenzene.

Results and discussion

Positive control results:
2,4-dinitro chlorobenzene at a concentration of 1% (w/w) induced positive skin sensitization reactions in 75% of the guinea pigs (ie 15 animals out of 20).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.1% + 100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0.1% + 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
0.1% + 100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.1% + 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.1% + 100%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0.1% + 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
0.1% + 100%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1% + 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
No cutaneous reactions were observed. According to the criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations) on the approximation of laws, regulations and administrative provisions relative to the classification, packaging and labelling of dangerous substances, the test item is not considered as a skin sensitizer .
Executive summary:

The potential of the test item 2,2'-AZOBIS(ISOBUTYRONITRILE) to induce skin sensitization was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman described in OECD guideline 406. The study was conducted in compliance with the principles of the Good Laboratory Practice Regulations.

Methods:

30 guinea-pigs were allocated to two groups: a control group1 (5 males and 5 females) and a treated group 2 (10 males and 10 females). On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed in the dorsal region between the shoulders. On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10% w/w) in order to induce local irritation. On day 8, this same test site was treated by topical application of the test substance (treated group) or the vehicle (control group) and was covered by an occlusive dressing for 48 hours. On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a topical application of the test substance to the right flank . The left flank served as a control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours. Skin reactions were evaluated approximately 24 and 48 hours later.

The test substance concentrations for induction were 0.1% (w/w) in paraffin oil and 100% for intradermal injections and topical application respectively. For the challenge application ,the test substance was undiluted for all groups.

The sensitivity of the guinea-pigs in the experimental conditions were checked in a study with positive sensitizer : 2,4 -dinitro chlorobenzene. During induction period, the positive sensitizer was applied at 0.1% (day 1) and 1% (day 8) concentrations. At cutaneous challenge application, 1%(w/w) was tested on the right flank.

Results:

No clinical signs and no treatment-related deaths were noted during the study. No cutaneous reactions were observed after the challenge application. The guinea-pigs used in a control study, showed a satisfactory sensitization response in 75% animals using the positive sensitizer.

Conclusion:

According to the maximisation method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance 2,2'-AZOBIS(ISOBUTYRONITRILE) were observed in guinea-pigs.

According to the criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations), the test item 2,2'-AZOBIS(ISOBUTYRONITRILE) is not classified as a sensitizer.