Reaction mass of sodium 3-[[4-[(4-ethoxy-m-tolyl)azo]-1-naphthyl]azo]benzenesulphonate and sodium 3-[[4-[(4-ethoxyphenyl)azo]-1-naphthyl]azo]benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Study period:

From May 30 to June 30, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted July 17, 1992

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study already available

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

In vivo test system

Test animals

Species:

guinea pig

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Test system: Ibm: GOHI; SPF-quality guinea pigs.
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 5 - 7 weeks.
- Weight at study initiation: 304 - 429 g; in pretest 331 - 400 g
- Housing: individually in Makrol on type-3 cages with autoclaved standard softwood bedding.
- Diet: pelleted standard Kliba 342, Batch no. 62/94 (at delivery of the animals to 02-JUN-1994) and 63/94 (from 03-JUN-1994 to termination of test) guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water.
- Acclimation period: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 25 °C
- Humidity: 56 - 82 %
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark.
- Other: music during the light period.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

5 %

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50 %

Challenge

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

25 %

No. of animals per dose:

Control Group 10 animals
Test Group 20 animals
Intracutaneous pretest 2 animals; epicutaneous pretest 4 animals

Details on study design:

TEST ARTICLE PREPARATION
The test article and the vehicles were placed into a glass beaker on a tared Mettler PM 480 balance and weight/weight dilutions were prepared. Homogeneity of test article in bi-distilled water and FCA/ physiological saline (1/1) was reached using a magnetic stirrer and a spatula was used for the test article in vaselinum album. The preparations were made immediately prior to each dosing.

RANGE FINDING TESTS
- Intradermal injections: 0.1 ml/site
- Concentrations: 5, 3 and 1% of the test article in bi-distilled water.
- No of animals: 2
- Observation: the resulting dermal reactions were assessed 24 hours later.

- Epidermal application: 4 patches* of filter paper (2 x 2 cm) were saturated with the test article at A = 50 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 25 %, C = 15 % and D = 10 % of the test article in vaselinum album and applied to the clipped and shaved flanks.
- Bendage: the patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape.
- No of animals: 4
- Exposure period: the dressings were removed after an exposure period of 24 hours.
- Observation: 21 hours after removing of the dressing the application site was depilated with an approved depilatory cream to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.
- Observations: the reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize criteria.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections / performed on test day 1
- Amount: 0.1 ml/site
- No. of exposures: three pairs of intradermal injections.
- Test groups: 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline; the test article, diluted to 5 % with bi-distilled water; the test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Control group: 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline; bi-distilled water; 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Site:

Epidermal applications / performed on test day 8
- Application: a 2 x 4 cm patch of filter paper was saturated with the test article (50 % in vaselinum album) and placed over the injection sites of the test animals.
- Bendage: the patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape.
- Exposure period: the dressings were left in place for 48 hours.
- Observations: reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

B. CHALLENGE EXPOSURE / performed on test day 22
- Days after induction: the test and control guinea-pigs were challenged two weeks after the epidermal induction application.
- Application: two patches ( 2 x 2 cm) of filter paper were saturated with the non-irritating concentration of 25 % (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application.
- Exposure period: the dressing were left in place for 24 hours.
- Observations: 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

INTERPRETATION
The results obtained from test animals following the challenge applications were compared with the results seen in control animals.
An allergic reaction was defined by visible reddening of the challenge site.
If the dermal reactions of test animals following the challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity.
If the dermal reactions of test animals following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive".
The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical to or less marked and/or persistent than the reactions observed in the control animals.

OBSERVATIONS
- Viability/Mortality: daily during the observation period.
- Clinical Signs (local/systemic): daily during the observation period.
- Skin reactions: at the times specified during the induction and challenge periods.
- Body Weights: at the beginning of the acclimatization period, at day one and at the termination of the test.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazol and 4-Aminobenzoic acid ethyl ester

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25 %

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

2-mercaptobenzothiazol

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25 %

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

4-Aminobenzoic acid ethyl ester

Any other information on results incl. tables

MAIN TEST

SKIN EFFECTS AFTER INTRADERMAL INDUCTION PERFORMED ON TEST DAY 1

CONTROL GROUP

- Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 6 and 2 to 5 respectively and then became necrotic from test day 7 to 11 followed by encrustation and exfoliation of encrustation up to test day 25 (termination of test).

- Injection site 2 (bi-distilled water)

The area around the injection site was oedematous and erythematous from test day 2 to 4.

- Injection site 3 (1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 1 with the mixture of FCA and physiological saline.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

TEST GROUP

- Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 4 and and then became necrotic from test day 5 to 7 followed by encrustation and exfoliation of encrustation up to test day 25.

- Injection site 2 (5% dilution of test article in bi-distilled water)

The area around the injection site was oedematous and orange discolored from test day 2 to 4 and 2 to 6 respectively, necrotic from test day 5 to 7. Encrustation was observed from test day 8 to 15 followed by exfoliation of encrustation up to the termination of test.

- Injection site 3 {5% dilution of test article in a 1:1 (v/v) mixture of FCA and physiological saline)

The area around the injection site was oedematous and orange discolored from test day 2 to 4 and 2 to 6 respectively, necrotic from test day 5 to 7. Encrustation was observed from test day 8 to 11 followed by exfoliation of encrustation up to the termination of test. As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

CONTROL GROUP

No erythematous or oedematous reaction was observed in the animals treated with vaselinum album only.

TEST GROUP

As the test article stained the skin orange, it was not possible to determine whether erythema was present. However, no oedema was observed. Orange discolouration was noted from test day 10 to 24.

All the animals were pretreated with a 10 % SLS in paraffinum perliquidum.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL GROUP

No positive reactions were observed in the animals either when treated with vaselinum album alone or when treated with the test article at 25 % in vaselinum album.

Orange discolouration was noted from test day 23 (after removal of the dressing to 24 (prior to the depilation).

TEST GROUP

No positive reactions were observed in the animals treated with vaselinum album.

When treated with the test article at 25% in vaselinum album, one animal exhibited a very slight erythematous reaction at the 24-hour reading.

Orange discolouration was noted from test day 23 (after removal of the dressing to 24 (prior to the depilation).

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

The body weight of the animals was within the normal range of variability.

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Not skin sensitising

Executive summary:

To assess the allergenic potential of test item in albino guinea pigs, the Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used. Ten males were used as control group and 20 males were used as test group. The highest non-irritating test article concentration used for challenge application was 25 % in vaselinum album.

In the study, 5 % of the animals of the test group were positive after treatment with a non-irritant test substance concentration of 25 % in vaselinum album. No positive reactions were observed in the control group.

The response of at least 30 % positive animals is considered positive; therefore the test article should be considered to be a non-sensitizer when described under the test conditions.

Conclusion

Not skin sensitising