Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

In vivo test performed

Type of information:

other: read-across from supporting substance (similar structure)

Adequacy of study:

key study

Study period:

From August 10, 1994 to September 12, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

see section any other materials and methods

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

see section any other materials and methods

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

LLNA method was not available in that period.

Test material

Test material information

Constituent 1

Reference substance name:

Similar substance 1

IUPAC Name:

Similar substance 1

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS- Source: CHARLES RIVER- Age at study initiation: 5 - 7 weeks- Weight at study initiation: Control and Test Group 291 - 356 g Pretest 282 - 334 g- Housing: Makrolon type-3 cages (size: 22x37x15 cm) with autoclaved standard softwood bedding- Diet: Pelleted standard Kliba 342, Batch no. 64/94 guinea pig breeding/ maintenance diet, 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. One day for the animals of the pretest.ENVIRONMENTAL CONDITIONS- Temperature (°C): 21 - 24.5 °C- Humidity (%): 56 - 86 %- Air changes (per hr): Air-conditioned with 10-15 air changes per hour- Photoperiod (hrs dark / hrs light): 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 and epicutaneous

Vehicle:

no data

Induction 2

Concentration / amount:

PRETESTINTRADERMAL INJECTIONS: Test group: 5, 3 and 1 % of the test article in bi-distilled water.EPIDERMAL APPLICATIONS:A = 25 % B = 15 %,C = 10 % D = 5 % MAIN STUDYINTRADERMAL INJECTIONS: Test group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test article, diluted to 5 % with bi-distilled water3) 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) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) Bi-distilled water3) 1:1 (w/w) mixture of in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.EPIDERMAL APPLICATION:Test group: 25 % in bi-distilled waterControl group: The guinea-pigs of the control group were treated with bi-distilled water only.CHALLENGE:Test group: 15 % with bi-distilled water

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

no data

Challenge 2

Concentration / amount:

PRETESTINTRADERMAL INJECTIONS: Test group: 5, 3 and 1 % of the test article in bi-distilled water.EPIDERMAL APPLICATIONS:A = 25 % B = 15 %,C = 10 % D = 5 % MAIN STUDYINTRADERMAL INJECTIONS: Test group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test article, diluted to 5 % with bi-distilled water3) 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) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) Bi-distilled water3) 1:1 (w/w) mixture of in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.EPIDERMAL APPLICATION:Test group: 25 % in bi-distilled waterControl group: The guinea-pigs of the control group were treated with bi-distilled water only.CHALLENGE:Test group: 15 % with bi-distilled water

No. of animals per dose:

NUMBER OF ANIMAL PER GROUP ANIMAL NUMBERS PER GROUP1 Control Group 10 346 - 3552 Test Group 20 356 - 3753 Intradermal Pretest 2 376 - 3774 Epidermal Pretest 4 378 - 381

Details on study design:

RANGE FINDING TESTS PRETEST: performed during the acclimatization period. The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the mainstudy. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.MAIN STUDY- No. of exposures: 3 intradermal injections- Test groups: 2 males guinea-pigs- Site: clipped flank- Concentrations: 0.1 ml/site of 5, 3 and 1% of the test article in bi-distilled water.- Result: For intradermal induction application a 5% test article dilution was selected.CHALLENGE EXPOSURE- No. of exposures: one application- Exposure period: The dressings were removed after an exposure period of 24 hours.- Test groups: 4 guinea pigs- Site: Both flanks - Concentrations: 4 patches of filter paper ( 2 x 2 cm) were saturated with the test article at: A = 25% (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 15%, C = 10% and D = 5% of the test article in bi-distilled water and applied to the clipped and shaved flanks.- Evaluation (hr after challenge): The dressings were removed after an exposure period of 24 hours. Approximately 21 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.- Result: For the epidermal induction the test article at 25% and for the challenge procedure the test article at 15% was selected.MAIN STUDY: Intradermal inductionA. INDUCTION EXPOSURE: performed on test day 1- No. of exposures: Three pairs of intradermal injections - Test groups: 20 males guinea pig- Control group: 10 males Guinea pig- Site: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. The injections were made at the border of a 4 x 6 cm area in the clipped region- Frequency of applications: 3 applications- Concentrations: 0.1 ml/siteTest Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) The test article, diluted to 5% with bi-distilled water3) 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) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.2) Bi-distilled water3)1:1 (w/w) mixture of in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.MAIN STUDY: Epidermal applicationsA. INDUCTION EXPOSURE: performed on test day 8, one week after the injections.- No. of exposures: one application- Exposure period: 48 hours.- Test groups: 20 male Guinea pigs- Control group: 10 males Guinea pigs- Site: scapular area (approximately 6 x 8 cm) was again clipped and shaved- Frequency of applications: one- Duration: 48 hours- Concentrations:Test group: 25% of test item in bi-distilled water and placed over the injection sites of the test animals. Control group: the guinea-pigs of the control group were treated with bi-distilled water only. 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- No. of exposures: one application- Day(s) of challenge: performed on test day 22- Exposure period: The dressing were left in place for 24 hours.- Test groups: 20 males Guinea pig- Control group: 10 males Guinea pig- Site: Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application.- Concentrations: 15% of teste item with bi-distilled water- Evaluation (hr after challenge): 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.OTHER:

Challenge controls:

The challenge site was evaluated 24 and 48 hours after the removal of the patch. The readings were made under artificial fluorescent light (daylight spectrum). Redness constitutes the minimum criterion of an allergic reaction. Strongly sensitized animals display a vivid redness, associated with indurated swelling. The reactions were scored on the basis of the Draize score described under "Readings and Scoring".

Positive control substance(s):

yes

Remarks:

4-AMINOBENZOIC ACID ETHYL ESTER and 2-MERCAPTOBENZOTHIAZOL

Results and discussion

Positive control results:

ran from 25-MAY- to 27-JUN-1994 and from 25-MAY- to 05-JUL-1994respectively. See Appendix D.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15% IN BI-DISTILLED WATER

No. with + reactions:

3

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 15% IN BI-DISTILLED WATER. No with. + reactions: 3.0. Total no. in groups: 20.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15% IN BI-DISTILLED WATER

No. with + reactions:

3

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 15% IN BI-DISTILLED WATER. No with. + reactions: 3.0. Total no. in groups: 20.0.

Any other information on results incl. tables

CONTROL GROUP

Intradermal induction:

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 from test day 2 to 4 and erythematous from test day 2 to 3, and became necrotic from test day 5 to 8. Encrustation was observed from test day 10 to 14 and exfoliation of encrustation from test day 15 to 25.

Injection site 2: (Bi-distilled water)

No erythematous or oedematous reaction was observed.

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.

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 from test day 2 to 4 and erythematous from test day 2 to 3, and became necrotic from test day 5 to 8. Encrustation was observed from test day 10 to 14 and exfoliation of encrustation from test day 15 to 27 (termination of test).

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

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

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 from test day 2 to 4 and erythematous from test day 2 to 3, and became necrotic from test day 5 to 8. Encrustation was observed from test day 10 to 14 and exfoliation of encrustation from test day 15 to 27 (termination of test). Oedematous reactions were again observed from test day 23 to 27.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL GROUP

Challenge:

No positive reactions were observed in the animals either when treated with bidistilled water alone or when treated with the test article at 15% in bidistilled water.

Brown discolouration was observed on test day 23 (after removal of the dressing) and 24 (prior to the depilation).

TEST GROUP

Challenge:

No positive reactions were observed in the animals treated with bi-distilled water alone.

Three animals were observed with very slight to well defined erythematous reactions at the 24- and 48-hour reading when treated with the test article at 15 % in bi-distilled water.

Brown discolouration was observed on test day 23 (after removal of the dressing) and 24 (prior to the depilation).

MORTALITY:

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

CLINICAL SIGNS:

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.

TEST GROUP: 6: SKIN RESPONSE AFTER THE CHALLENGE PROCEDURE

Animal number	Sex	24 hours		48 hours
		Erythema	Oedema	Erythema	Oedema
356	male	0	0	0	0
357	male	2	0	2	0
358	male	0	0	0	0
359	male	2	0	2	0
360	male	0	0	0	0
361	male	0	0	0	0
362	male	0	0	0	0
363	male	1	0	1	0
364	male	0	0	0	0
365	male	0	0	0	0
366	male	0	0	0	0
367	male	0	0	0	0
368	male	0	0	0	0
369	male	0	0	0	0
370	male	0	0	0	0
371	male	0	0	0	0
372	male	0	0	0	0
373	male	0	0	0	0
374	male	0	0	0	0
375	male	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to the CLP Regulation (EC n. 1272/2008)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The substance is not sensitiser

Executive summary:

The Similar Substance 1 has been tested for skin sensitisation according to the Maximization-Test of B. Magnusson and A.M. Kligman (1969).

The purpose of this skin sensitization study was to assess the allergenic potential of test item when administered to the skin of albino guinea pigs.

This study should provide a rational basis for risk assessment of the sensitizing potential of the test article in man.

The sensibility and reliability of the experimental technique used was assessed by use of substances which are known to have skin sensitization properties in the guinea pig strain. The positive controls were performed with 4-AMINOBENZOIC ACID ETHYL ESTER and 2 -MERCAPTOBENZOTHIAZOL.

Erythema and oedema were assessed using the following numerical grading system according to Draize.

The pretest was performed during the acclimatization period.

The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.

In the main study the test article was diluted to 5% with bi-distilled water for intradermal induction and the 25% of test item in bi-distilled water were placed over the injection sites of the test animals.

For challenge induction the 15% of test item was used as highest non-irritating concentration.

CONCLUSION

In this study 15 % of the animals were positive after treatment with a non irritant test substance concentration of 15 % in bi-distilled water.

The response of at least 30 % positive animals is considered positive following the CLP Regulation (EC n. 1272/2008)

Therefore the test article is not considered to be a sensitizer when described under the test conditions.