3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

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:

September 03, 2001 to November 16, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

July 30, 1996

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)

GLP compliance:

yes

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: RCC Ltd, Biotechnology & Animal Breeding Division, W61ferstrasse 4, CH-4414 FOllinsdorf I Switzerland
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-quality (Specific Pathogen Free)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: Body weight at pretest start: Pretest groups: 389 - 404 g. Body weight at beginning of acclimatization period: Control and test group: 335 - 394 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418, batch no. 91 /01, guinea pig breeding / maintenance diet, containing Vitamin C (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum. Results of analyses for contaminants are archived at RCC Ltd, ltingen.
- Water (e.g. ad libitum): Community tap water from Fullinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd, ltingen.
- 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 (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From delivery: 03 September 2001 (pretest) / 10 September 2001 (main test) To: 11 October 2001 (termination)

Route:

intradermal

Vehicle:

water

Concentration / amount:

10%

Day(s)/duration:

Day 1

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

15%

Day(s)/duration:

Day 8

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

5%

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

PRETEST: 1 for intradermal pretest; 2 for epidermal pretest
MAIN TEST: 5 in control group; 10 in test group

Details on study design:

RANGE FINDING TESTS:

a) 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 (no. 81 ). One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of A = 10 %, B = 5 % and C = 3 % of the test item in bidistilled water. The three concentrations were determined during non-GLP formulation trials performed prior to the pretest. The concentration of 10 % was considered to be the highest technically applicable concentration which could be injected into the intra-cellular space in
spite of the high viscosity of the application dilution and the obstacle caused by the tissus.
Dermal reactions were assessed 24 hours later.
Based on the results, the test item concentration of 1 O % was selected for intradermal induction in the main study.

b) EPIDERMAL APPLICATIONS:
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 two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (3 x 3 cm) were saturated with the test item at D = 15 % (technically the highest possible concentration to be applied sufficiently), E = 10 %, F = 5 % and G = 1 % in bi-distilled water and applied to the clipped and shaved flanks. The amount of test item preparation applied was approximately 0.2 g for the test item
at 15 % and a volume of approximately 0.2 ml was applied for the remaining test item 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 item. 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) 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. The position of the epidermal applications is as follows:

Animal n°82: D -> left cranial, E -> left caudal; F -> right cranal; G -> right caudal
Animal n°83: G -> left cranial, D -> left caudal; E -> right cranal; F -> right caudal

The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness. Based on the results obtained the concentration selected for induction and challenge in the main study was 15 % and 5 %, respectively.

MAIN STUDY
A. INDUCTION 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 item, at 10 % in bi-distilled water.
3) The test item at 10 % 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 item (15 % in bi-distilled water) and placed over the injection sites of the test animals. The amount of test item 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 ensured intensive contact of the test item.
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 (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 item at the highest tested non-irritating concentration of 5 % (applied to the 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 item 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 item 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.

C) Interpretation
The results obtained from test animals following the challenge application 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 or less marked and/or persistent than the reactions observed in the control animals.
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 may not necessarily be due to allergenicity, but instead may be a false-positive irritant response caused by 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 item 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 from delivery of the animals to the termination of the test.
Clinical signs (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 1 and termination of the test.

F) NECROPSY
Necropsy was performed in one animal (no. 91) of the test group which was found dead on test day 24 (i.e. day of the 24-hour reading in the challenge phase).
No necropsies were performed in the animals of the control and test group sacrificed at termination of the observation period nor in the animals of the intradermal and epidermal pretest sacrificed on test day 1 of the main study.
The surviving animals were sacrificed by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of at least 2.0 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight) and discarded.

STATISTICAL ANALYSIS
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.

Challenge controls:

5 animals

Positive control substance(s):

no

Remarks:

Not tested in the current sudy. The sensitivity and reliability of the experimental technique employed was assessed by use of 2-MERCAPTOBENZOTHIAZOLE. The results from the most recent test run are available in the study report.

Positive control results:

For validation of sensitivity of the Maximization-Test (m&K) 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 TG 406, adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993).
The raw data from this project are kept in a separate file at RCC Ltd.
The study was performed with 15 (10 test and 5 control) male albino guinea pigs (GOHI), delivered by RCC Ltd, Biotechnology & Animal Breeding Division (CH-4414 FOllinsdorf / Switzerland).
The intradermal induction of sensitization in the test group was performed in the nuchal region with a 5 % dilution of the test item in mineral oil and in an emulsion of FCA/ physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the test item at 50 % in mineral oil one week after the intradermal induction. The animals of the control group were intradermally induced with mineral oil and FCA/physiological saline and epidermally induced with mineral oil under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 0.5 % in mineral oil and mineral oil 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.
All 10 test animals showed discrete/patchy to intense erythema and swelling at the 24- and 48-hour reading after the challenge treatment with 2-MERCAPTOBENZOTHIAZOLE at 0.5 % (w/w) in mineral oil.
No skin effect was observed in the control group.

Based on the findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC, 2-MERCAPTOBENZOTHIAZOLE does have to be classified and labelled as a skin sensitizer.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Induction with water only. Challenge: water only (left flank) and test item at 5% (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were observed in the animals. Blue discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Induction with water only. Challenge: water only (left flank) and test item at 5% (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were observed in the animals. Blue discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induction with test item (10% in water). Challenge: water only (left flank) and test item at 5% (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were observed in the animals. Blue discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induction with test item (10% in water). Challenge: water only (left flank) and test item at 5% (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions were observed in the animals. Blue discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Remarks on result:

no indication of skin sensitisation

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

One animal (no. 91) of the test group was found dead on test day 24 (i.e. day of the 24-hour reading in the challenge phase). At necropsy, no macroscopic findings were noted. The

cause of death could not be established. The death was considered to be spontaneous and treatment unrelated.

CLINICAL SIGNS, SYSTEMIC

No signs of systemic toxicity were observed in the animals.

BODY WEIGHTS

Animals no. 84 of the control group and nos. 89, 98 of the test group showed a loss of body weight (1.8 to 3.9 %) during the acclimatization period. They 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.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on criteria defined in Regulation (EC) No 1272/2008, BLUE GS 5664.80 is considered to be "not sensitzing" to guinea pigs and is not classified according to CLP criteria.

Executive summary:

In order to assess the cutaneous allergenic potential of BLUE GS 5664.80, the Maximization-Test was performed in 15 (10 test and 5 control) female 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 10 % dilution of the test item 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 item at 15 % in bi-distilled 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 bidistilled water under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 5 % 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.

Skin reactions 24 and 48 hours after the challenge procedure were nil in control (5 animals) and test groups (9 animals) on leftt (test item, 5% in water) and right (water only) flanks with 0% of positive responses.

Note that one animal of the test group was found dead on test day 24 (i.e. day of the 24-hour reading in the challenge phase). At necropsy, no macroscopic findings were noted. The cause of death could not be established. The death was considered to be spontaneous and treatment unrelated.

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

None of the control and test animals showed skin reactions after the challenge treatment with BLUE GS 5664.80 at 5 % (w/w) in bi-distilled water.

Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC and Regulation (EC) No 1272/2008 (CLP), BLUE GS 5664.80 does not have to be classified and labelled as a skin sensitizer.

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,  BLUE GS 5664.80 is considered to be "not sensitzing" to guinea pigs and is not classified according to CLP criteria.