disodium 7-(4,6-difluoropyrimidin-2-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate; reaction mass of: disodium 7-(2,4-difluoropyrimidin-6-ylamino)-4-hydroxy-3-(4-methoxy-2-sulfonatophenylazo)naphthalene-2-sulfonate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Delivery of animals: October 23, 1996 - Termination: December 03, 1996 - Reported: January 07, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

July 31, 1992

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

As specified in ECHA guidance R7a (v5.0 - December 2016):
"For new in vivo testing of skin sensitisation potential, the murine local lymph node assay (LLNA), which is currently the best in vivo method to assess skin sensitisation potency, is the REACH Annex VII-endorsed in vivo method."
"Two further animal test methods for skin sensitisation are described in EU B.6/OECD TG 406: the guinea pig maximisation test (GPMT) and the Buehler test."
"Both the GPMT and the Buehler tests are able to detect substances with moderate to strong sensitisation potential, including those with relatively weak sensitisation potential."
"Since the LLNA is the preferred method for new in vivo testing, the use of the standard guinea pig tests to obtain new data on the skin sensitisation potential of a substance will be acceptable only in exceptional circumstances and will require scientific justification. However, existing data of good quality that were generated before 11 October 2016, or for which the study was initiated before 11 October 2016, and derived from such tests are acceptable; and if these tests provide clear results that are adequate for classification, even when a conclusion on potency (Cat. 1A or not) cannot be drawn, they will preclude the need for further in vivo testing."
The current study is a GMPT test performed before 11 October 2016 and providing clear results.

Species:

guinea pig

Strain:

other: lbm: GOHI; SPF-quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, 4414 FOllinsdorf / Switzerland
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-quality (Specific Pathogen Free)
- Age at study initiation (at delivery): 5-7 weeks
- Weight at study initiation:
Body weight at delivery: pretest 290-313 g
Body weight at beginning of Acclimatization period - control and test group : 289-463 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Nafag Ecosan 845 25W4, batch nos. 90/96, 105/96 and 118/96 guinea pig breeding/ maintenance diet ("Nafag", N~hr- und Futtermittel AG, CH- 9202 Gossau), ad libitum. Results of analysis for contaminants are included in the study report.
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/l) via the drinking water. Results of bacteriological, chemical and contaminant analyses are included in the study report.
- 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.
- Indication of any skin lesions: none

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70, (values above 70 % during cleaning process possible)
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12, music during the light period.

- IN-LIFE DATES: From delivery: Delivery of the animals 23-0CT-1996, Pretest 23-0CT-1996 to 06-NOV-1996, acclimatization 30-0CT-1996 to 05-NOV-1996, treatment/observation 06-NOV-1996 to 03-DEC-1996, termination 03-DEC-1996.

Route:

intradermal

Vehicle:

water

Concentration / amount:

The intradermal induction of sensitization was carried out with a 5 % dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA)/physiological saline

Day(s)/duration:

Day 1/ 24 hours

Route:

epicutaneous, occlusive

Vehicle:

water

Day(s)/duration:

Day 8/ 48h

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

Two weeks after the epicutaneous induction application the
challenge was completed by epicutaneous application of the test article at 10%
in bi-distilled water under occlusive dressing.

Day(s)/duration:

Day 22/48h

No. of animals per dose:

CONTROL GROUP: 10 animals
TEST GROUP: 20 animals
INTRADERMAL PRETEST: 2 animals
EPIDERMAL PRETEST: 4 animals

Details on study design:

RANGE FINDING TESTS:

a) INTRADERMAL INJECTIONS:
Two pairs of intradermal injections (0.1 ml /site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline was made simultaneously into the shaved neck of two guinea pigs. One week later intradermal injections (0.1
ml/site) were made into the clipped flank of the same guinea pigs at concentrations of 5, 3 and 1 % of the test article in bi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5% test article dilution was selected.

b) EPIDERMAL APPLICATIONS:
Two pairs of intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline was made simultaneously into the shaved neck of four guinea pi gs. One week 1 ater both flanks of each of the
guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A= 50%, B = 25%, C = 15% and D = 10% in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied was approximately ~ 0.2 ml. 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 (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.
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. The animals were then 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 on a numerical basis according to Draize described above.

MAIN STUDY:

a) IInduction exposure

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, diluted to 5% with bi-distilled water
3) 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 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 clipped and shaved free of hair. 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 volume of test article applied was approximately 0.3 ml. 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 dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea pigs of the control group were treated as described above with bidistilled 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 (performed on test day 22)
The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs 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 (2 x 2 cm) of filter paper were saturated with 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 the test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages.
Twenty-four 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.

C) 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. W
By "maximizing" the exposure and enhancing allergenicity, some problems could arise, particularly in relation to specificity, especially the potential for false-positive reactions . An inflammatory response at challenge does not automatically bespeak allergenicity, but instead may be a false-positive irritant response from an inducing hyperirritability .

D) RATING OF ALLERGENICITY ACCORDING TO MAGNUSSON AND KLIGMAN
Based upon the percentage of animals sensitized (24- and 48-hour reading), the test article was assigned to one of the following five grades of allergenic potency according to Magnusson and Kligman, ranging from weak to extreme.
Sensitization rate between 0 and 8% -> grade 1 --> classification as weak
Sensitization rate between 9 and 28% -> grade 2 --> classification as mild
Sensitization rate between 29 and 64% -> grade 3 --> classification as moderate
Sensitization rate between 65 and 80% -> grade 4 --> classification as strong
Sensitization rate between 81 and 100% -> grade 5 --> classification as extreme

E) Observations
The following observations and data were recorded during the study:
Viability / Mortality: daily during the observation period
Clinical signs (local/systemic): daily during the observation period
Skin reactions: at the ti mes specified during the induction and challenge periods.
Body weights: at delivery of the animals (pretest), beginning of the accl i mati zati on peri ad (main study), day one and termination of the test.

F) NECROPSY
Necropsy was performed by an experienced prosector in one animal of the test group found dead on test day 8 and in one animal of the control group euthanized on test day 22.
No necropsies were performed in the animals sacrificed at termination of the observation period.
The surviving animals were sacrificed by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of at least 5.1 ml/kg body weight (equivalent to 810 mg sodium pentobarbitone/kg body weight) and discarded.

STATISTICAL ANALYSIS
Mean values with standard deviations are described in the body weight tables.

Challenge controls:

10 animals

Positive control substance(s):

yes

Remarks:

2-MERCAPTOBENZOTHIAZOLE

Positive control results:

For validation of sensitivity of the Maximization-Test of B. Magnusson and A.M. Kligman (1969) as well as the sensitivity of the test system used, a known sensitizer 2-MERCAPTOBENZOTHIAZOLE was selected as a positive control. This was performed in accordance with the recommendation of the OECD for testing of chemicals number 406 "Skin Sensitization Test", adopted by the council on July 17, 1992.

RESULTS
The non-irritating test article concentration used for challenge application was 50 % in mineral oil.
According to the procedures used in this experiment (performed from 30-APR-1996 to 07-JUN-1996) positive results were observed in the treated animals after the epidermal challenge application.
In this study 90 % (at the 24-hour reading) ahd 100 % (at the 48-hour reading) of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 50 % in mineral oil. No skin reactions were observed in the control group.

A response of at least 30 % positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 93/21/EEC, April 27, 1993 adapting to technical progress for the 18th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances".

Therefore, the test article 2-MERCAPTOBENZOTHIAZOLE applied at a concentration
of 50 % in mineral oi 1 is considered to be a sensitizer when used under the described test conditions.

According to the rating of allergenicity by Magnusson and Kligman the test article is an extreme sensitizer.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Key result

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

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

One animal of the control group (no. 669) was euthanized on test day 22, since a paresis of the left and right hindlegs was observed. One animal of the test group (no. 672) was found dead on test day 8, prior to the epidermal induction application. At necropsy, a reddish discoloration of the lungs was noted.

CLINICAL SIGNS, SYSTEMIC

No signs of systemic toxicity were observed in the animals.

BODY WEIGHTS

The body weight of the animals was within the range of physiological variability known for this strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on criteria defined in Regulation (EC) No 1272/2008, SCARLET RN 1165 is considered to be "not sensitzing" to guinea pigs and is not classified according to CLP criteria.

Executive summary:

SUMMARY

In order to assess the cutaneous allergenic potential of SCARLET RN 1165, the Maximization-Test in accordance with OECD Guideline No. 406 and the Directive 92/69 EEC B.6 was carried out on 30 (20 test and 10 control) female albino guinea pigs.

The intradermal induction of sensitization was carried out with a 5 % dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA)/physiological saline. The epicutaneous induction of sensitization was conducted under occlusion with the test article at 50% in bidistilled water. Two weeks after the epicutaneous induction application the challenge was completed by epicutaneous application of the test article at 10% in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological saline and challenged

similarly as those of the test group. Cutaneous reactions, i.e erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.

ERYTHEMATOUS REACTIONS AFTER THE FIRST CHALLENGE PROCEDURE

	after 24 hours	After 48 hours
	(Positive/total)/% positive of total
CONTROL GROUP
SCARLET RN 1165 (left flank)	(0/9*)/0	(0/9*)/0
Bi-distilled water only (right flank)	(0/9)/0	(0/9*)/0
TEST GROUP
SCARLET RN 1165 (left flank)	(1/19*)/5	(1/19)/5
Bi-distilled water only (right flank)	(0/19)/0	(0/19)/0

* One animal of the control group (no.669) was euthanized on test day 22 due to ethical reasons and one animal of the test group (no.672) was found dead on test day 8. The death occurred in the test group was considered to be treatment unrelated since neither clinical signs nor deaths were observed in any other animal.

No toxic symptoms were evident in the guinea pigs of the control or test group.

CONCLUSION

In this study 5% of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 10% in bi-distilled water. No skin reactions were observed in the control group.

Therefore, the test article SCARLET RN 1165 applied at a concentration of 10% in bi-distilled water is considered to be a non-sensitizer when used under the described test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on criteria defined in Regulation (EC) No 1272/2008, the test item is considered to be "not sensitzing" to guinea pigs and is not classified according to CLP criteria.