2,2'-azobis[2-methylpropionamidine] dihydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 01 - October 25, 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Batchno and purity were not given, and limited reported housing conditions.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

study was already available before entry into force of REACH

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

EC Number:

221-070-0

EC Name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

Cas Number:

2997-92-4

Molecular formula:

C8H18N6.2ClH

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylpropanimidamide) dihydrochloride

Details on test material:

- Name of test material (as cited in study report): V-50

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Dean Dual, Luxemburg, Wisconsin
- Age at study initiation: young adults
- Weight at study initiation: 331-421 g
- Housing: Individually in stainless-steel cages
- Diet (e.g. ad libitum): Continuous access to Purina Guinea Pig Chow
- Water (e.g. ad libitum): Continuous access to water
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data, temperature-controlled rooms
- Humidity (%): no data, humidity-controlled rooms
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: 01 October 1981 To: 22 October 1981

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

petrolatum

Induction 2

Concentration / amount:

The Freund's Complete Adjuvant Solution used in all intradermal injections was prepared by adding 2.5 ml of sterile water for injection in approximately 1.0 ml increments to 2.5 ml of Freund's Adjuvant.

The 5% solution of test material in sterile water was prepared by taking 0.5g of V-50 and adding sterile water for injection to a total volume of 10.0 ml.

The 5% solution of test material in Freund's Adjuvant was prepared by adding 2.5 ml of a 10% w/v mixture of V-50 in sterile water to 2.5 ml of Freund's Adjuvant solution. The test material was added in approximately 1.0 ml increments.

For the topical induction and challenge procedure, the test material was administered as 50% w/w and 25% w/w mixtures, respectively, in petrolatum.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Challenge 2

Concentration / amount:

The Freund's Complete Adjuvant Solution used in all intradermal injections was prepared by adding 2.5 ml of sterile water for injection in approximately 1.0 ml increments to 2.5 ml of Freund's Adjuvant.

The 5% solution of test material in sterile water was prepared by taking 0.5g of V-50 and adding sterile water for injection to a total volume of 10.0 ml.

The 5% solution of test material in Freund's Adjuvant was prepared by adding 2.5 ml of a 10% w/v mixture of V-50 in sterile water to 2.5 ml of Freund's Adjuvant solution. The test material was added in approximately 1.0 ml increments.

For the topical induction and challenge procedure, the test material was administered as 50% w/w and 25% w/w mixtures, respectively, in petrolatum.

No. of animals per dose:

Treated group 15 animals, and a naive control group, positive control group and vehicle group of four animals each.

Details on study design:

RANGE FINDING TESTS: A range-finding study to determine the irritation threshold of the test material was conducted. Based upon these results, the test material was administered as 50% w/w mixture in petrolatum for the topical induction and as 25% w/w in petrolatum for the challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: two
- Exposure period: intradermal injections: 7 days, topical induction: 48 hours
- Test groups: 50% w/w mixture of V-50 in petrolatum
- Control group: naive control group, positive control group (0.1% w/v mixture of 2,4-Dinitrochlorobenzene in sterile water) and vehicle control group (sterile 0.9% saline)
- Site: A 4.0x6.0 com area was clipped along the midline across the shoulder region,
- Frequency of applications: one

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: two weeks after topical induction the animals received a challenge dose for 24 hours.
- Exposure period: 24 hours
- Test groups: 25% w/w mixture of V-50 in petrolatum
- Control group: positive control group and vehicle control group.
- Site: The hair was removed from a 5.0x5.0 cm area on the flank by clipping and as reported at the induction exposure.
- Evaluation (hr after challenge): 24 and 48 hours following patch removal the test sites were examined for erythema and edema.

Positive control substance(s):

yes

Remarks:

The 0.1% solution of 2,4-Dinitrochlorobenzene in sterile water was prepared by adding 0.10 g of 2,4-Dinitrochlorobenzene to 100 ml of sterile water. The solution was mildly heated and thoroughly stirred prior to use.

Results and discussion

Positive control results:

A sensitization reaction to the positive control was exhibited by all animals following the challenge application. All animals responded with a moderate and diffuse redness at 24 hours. At the 48 hour observation a moderate and diffuse redness was seen in one animal and three animals had a mild redness.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Challenge dose 25% w/w

No. with + reactions:

13

Total no. in group:

15

Clinical observations:

Ten animals reacting to the challenge application exhibited a moderate and diffuse redness. Three animals exhibited a mild redness. Two test animals were found dead on Day 11 of the study.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Challenge dose 25% w/w. No with. + reactions: 13.0. Total no. in groups: 15.0. Clinical observations: Ten animals reacting to the challenge application exhibited a moderate and diffuse redness. Three animals exhibited a mild redness. Two test animals were found dead on Day 11 of the study..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Challenge dose 25% w/w

No. with + reactions:

13

Total no. in group:

15

Clinical observations:

Six animals continued to exhibited a moderate and diffuse redness while the remaining seven animals exhibited a mild response.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Challenge dose 25% w/w. No with. + reactions: 13.0. Total no. in groups: 15.0. Clinical observations: Six animals continued to exhibited a moderate and diffuse redness while the remaining seven animals exhibited a mild response..

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

other: untreated

Dose level:

Challenge dose 25% w/w

No. with + reactions:

3

Total no. in group:

4

Clinical observations:

Three animals responded to the challenge application with a mild redness.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: other: untreated. Dose level: Challenge dose 25% w/w. No with. + reactions: 3.0. Total no. in groups: 4.0. Clinical observations: Three animals responded to the challenge application with a mild redness..

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

other: untreated

Dose level:

Challenge dose 25% w/w

No. with + reactions:

1

Total no. in group:

4

Clinical observations:

A mild redness was observed in one animal.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: untreated. Dose level: Challenge dose 25% w/w. No with. + reactions: 1.0. Total no. in groups: 4.0. Clinical observations: A mild redness was observed in one animal..

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

other: vehicle control

Dose level:

Sterile 0.9% Saline

No. with + reactions:

0

Total no. in group:

4

Clinical observations:

None of the guinea pigsexhibited a reaction to the challenge dose.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: other: vehicle control. Dose level: Sterile 0.9% Saline. No with. + reactions: 0.0. Total no. in groups: 4.0. Clinical observations: None of the guinea pigsexhibited a reaction to the challenge dose..

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

other: vehicle control

Dose level:

Sterile 0.9% Saline

No. with + reactions:

0

Total no. in group:

0

Clinical observations:

None of the guinea pigs exhibited a reaction to the challenge dose.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: vehicle control. Dose level: Sterile 0.9% Saline. No with. + reactions: 0.0. Total no. in groups: 0.0. Clinical observations: None of the guinea pigsexhibited a reaction to the challenge dose..

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1% w/v Dinitrochlorobenzene

No. with + reactions:

4

Total no. in group:

4

Clinical observations:

All animals responded with a moderate and diffuse redness.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1% w/v Dinitrochlorobenzene. No with. + reactions: 4.0. Total no. in groups: 4.0. Clinical observations: All animals responded with a moderate and diffuse redness..

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1% w/v Dinitrochlorobenzene

No. with + reactions:

4

Total no. in group:

4

Clinical observations:

A moderate and diffuse redness was seen in one animal and three animals had a mild redness.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1% w/v Dinitrochlorobenzene. No with. + reactions: 4.0. Total no. in groups: 4.0. Clinical observations: A moderate and diffuse redness was seen in one animal and three animals had a mild redness..

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based upon the results obtained, the substance is considered a strong skin sensitizer in guinea pigs.
According to Guidance on information requirements and chemical safety assessment Chapter R.8, Appendix R.8-10, the substance is allocated to the moderate potency category.

Executive summary:

This study was designed to evaluate the skin sensitization properties of the substance using the Maximization test. The study consisted of a treated group of 15 animals, and a naive control group, positive control group (Dinitrochlorobenzene) and vehicle group (0.9% saline) of four animals each. In the test substance group, 0.05 ml test substance was injected intradermally on each side of the midline across the shoulder region of guinea pigs for the first induction. After 7 days, they were secondly induced with a 50% w/w mixture of V-50 in petrolatum for 48 hours. After two weeks, the right flank was challenged with 25% w/w test substance for 24 hours. Vehicle and positive control group were treated similarly. As a result of the study, all surviving animals of the test substance group and all animals of the positive control group exhibited a sensitization reaction following the challenge application. None of the animals exhibited a reaction in the vehicle control group. According to the results, it is concluded that the substance has strong skin sensitization properties in guinea pigs. Classification: Skin Sens. Cat. 1, May cause an allergic reaction (H317).