Tetrasodium 2-[[8-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]benzoyl]amino]-1-hydroxy-3,6-disulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

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 September 13 to October 21, 1999

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:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted on July 17, 1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Test performed on 1999.

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland.
- Age at study initiation: 4 - 6 weeks.
- Weight at study initiation: pretest groups 354 - 405 g; body weight at beginning of acclimatization period control and test group 309 - 398 g.
- Housing: individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Nafag Ecosan 845 25W4, batch no.79/99, guinea pig breeding / maintenance diet ("Nafag", Näihr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water: community tap water from Füllinsdorf, ad libitum.
- 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 visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 40 - 70 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: automatically controlled light cycle of 12 hours light and 12 hours dark. Fluorescent "Gold" lamps (Silvania Gold F40T1260) were present in the room used for the study. Music was played 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:

water

Concentration / amount:

50 %

Day(s)/duration:

2

Challenge

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

10 %

Day(s)/duration:

1

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Control group: 5 males
Test group: 10 males

Details on study design:

RANGE FINDING TESTS
- No of animals per dose: 1 animal intradermal pretest and 2 animals epidermal pretest.
INTRADERMAL INJECTIONS
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in bi-distilled water.
Dermal reactions were assessed 24 hours later. Based on the results, a test article concentration of 5 % was selected for intradermal induction in the main study.

EPIDERMAL APPLICATIONS
4 patches of filter paper (3 x 3 cm) were saturated with the test article at 50 % (this concentration was found to be the maximum possible under the conditions of the procedure), 25 %, 15 % and 10 % in bi-distilled water and applied to the clipped and shaved flanks. The amount of test article preparation applied was approximately 0.2 g for the test article at 50 Vo and a volume of approximately 0.2 ml was applied for the remaining test article concentrations. 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. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.
Twenty-one hours after removal of the dressing the application site was depilated with an approved depilatory cream in order to visualize any resulting erythema.
The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water.
Thereafter, the animals were dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

MAIN STUDY
INTRADERMAL INJECTIONS / PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in bidistilled water.
3) The test article at 5 % 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 water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS / PERFORMED ON TEST DAY 8
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test article (50 % in bi-distilled water) and placed over the injection sites of the test animals. The amount of test article preparation applied was approximately 0.3 g. 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. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensures intensive contact of the test article.
The guinea pigs of the control group were treated as described above with bi-distilled water only, applied at a volume of approximately 0.3 ml.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE
CHALLENGE / PERFORMED ON TEST DAY 22
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.
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. Two patches (3 x 3 cm) of filter paper were saturated with the test article at the highest non-irritating concentration of 10 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article preparation and vehicle applied was approximately 0.2 ml. The dressings were left in place for 24 hours.
Twenty-one hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

OBSERVATIONS
- Viability / Mortality: daily from delivery of the animals to the termination of the test.
- Clinical signs (local/systemic): daily from delivery of the animals to the termination of the test.
- Skin reactions: at the times specified during the pretest, induction and challenge periods.
- Body weights: at pretest and acclimatization start, day I and termination of the test.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

5

Total no. in group:

10

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

-

No. with + reactions:

0

Total no. in group:

10

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50 %

No. with + reactions:

10

Total no. in group:

10

Any other information on results incl. tables

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

CONTROL GROUP: no erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.

TEST GROUP: as the test article at 50 % stained the skin red-brown, it was not possible to determine whether erythema was present or not. However, no oedema was observed.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL GROUP: no skin reactions were observed in the animals when treated with either bi-distilled water only or when treated with the test article at l0 % in bi-distilled water. Red-brown discoloration was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

TEST GROUP: discrete/patchy to moderate/confluent erythema were observed in five out of 10 animals at the 24- and 48-hour reading after treatment with the test article at l0 % in bi-distilled water. No skin reactions were observed in the animals when treated with bi-distilled water only. Red-brown discoloration was noted directly after removal of the patch.

	After 24 hrs		After 48 hrs
	positive / total	% positive of total	positive / total	% positive of total
Control group
Test item 10 % in bi-distilled water	0 / 5	0	0 / 5	0
Bi-distilled water only	0 / 5	0	0 / 5	0
Test group
Test item 10 % in bi-distilled water	5 / 10	50	5 / 10	50
Bi-distilled water only	0 / 10	0	0 / 10	0

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

There were no deaths during the course of the study, hence no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

No signs of systemic toxicity were observed in the animals.

BODY WEIGHTS

Animal no. 982 of the test group showed a loss of body weight during the acclimatization period. It recovered between the treatment start and the end of the study.

The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens 1B (H317), according to the CLP Regulation (EC) No 1272/2008

Conclusions:

Skin sensitizer

Executive summary:

In order to assess the cutaneous allergenic potential of test item, the Maximization-Test was performed in 15 (10 test and 5 control) male albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5 % dilution of the test article in bi-distilled water and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test article at 50 % in bidistilled water one week after the intradermal induction. The animals of the control group were intradermally induced with bi-distilled water and FCA/physiological saline and epidermally induced with bi-distilled water under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test article at 10 % in bi-distilled water and bi-distilled water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. Five out of 10 test animals showed discrete/patchy to moderate/confluent erythema after the challenge treatment with test item at 10 % (wlw) in bi-distilled water. No skin effect was observed in the control group.

Conclusion

Based on the above mentioned findings in an adjuvant sensitization test in guinea pigs and in accordance to CLP Regulation (EC) No 1272/2008 has to be classified and labelled as a skin sensitizer, category 1B.