disodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[(4-vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; reaction mass of: trisodium 5-{4-chloro-6-[N-ethyl-(3-(2-sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate; tetrasodium 5-{4-chloro-6-[N-ethyl-3-(2-(sulfonatooxy)ethylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]-4-hydroxynaphthalene-2,7-disulfonate; trisodium 5-{4-chloro-6-[N-ethyl-3-(vinylsulfonyl)anilino]-1,3,5-triazin-2-ylamino}-4-hydroxy-3-[4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 09, 2002 - July 04, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

July 30, 1996

Test guideline 2

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.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-050-5

EC Name:

-

IUPAC Name:

dodecasodium 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-({4-chloro-6-[ethyl({3-[2-(sulfooxy)ethanesulfonyl]phenyl})amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-3-[(E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate 5-[(4-chloro-6-{[3-(ethenesulfonyl)phenyl](ethyl)amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(E)-2-{4-[2-(sulfooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid

Details on test material:

Identity: Red Rwa 4565
Appearance: Solid, red powder
Storage: at room temperature at about 20°C

In vivo test system

Test animals

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: 4-7 weeks
- Weight at study initiation: Body weight at pretest start: Pretest groups: 360-446 g. Body weight at beginning of acclimatization period: Control and test group: 365-433 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): 20 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From delivery: 09 April 2002 (intradermal and epidermal pretest I) / 23 April 2002 (epidermal pretest II) / 16 Apreil 2002 (main test) to 17 May 2002 (termination)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

10%

Day(s)/duration:

Day 1

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Day(s)/duration:

Day 8

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

3%

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 I; 2 for epidermal pretest II
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 AdjuvanVphysiological saline were made into the shaved neck of one guinea pig (no. 685). One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of A=10o/o, B= 5% and C=3% of the test item in purified water.
Dermal reactions were assessed 24 hours later.
Based on the results, the test item concentration of 10 7o 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 AdjuvanUphysiological 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 = 50 % (technically the highest possible concentration to be applied sufficiently), E=25o/o, F = 15 % and G = 1OT"in purified water and applied to the clipped and shaved flanks. The volume of test item preparation 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 imperuious 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. lt 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 (see 4.9). No highest non-irritating concentration could be determined after the epidermal pretest described above. Therefore, a second pretest was performed with two additional naive guinea pigs (nos. 7O3 - 7O4), treated in the same way as those described previously, with the concentrations of H=5%, I=3%, J=1% and K=0.5% in purified water, using an application volume of approximately 0.2 ml.
The position of the epidermal applications is shown below:

Animal n°686: D -> left cranial, E -> left caudal; F -> right cranal; G -> right caudal
Animal n°687: G -> left cranial, D -> left caudal; E -> right cranal; F -> right caudal
Animal n°703: H -> left cranial, I -> left caudal; J -> right cranal; K -> right caudal
Animal n°704: K -> left cranial, H -> left caudal; I -> right cranal; J -> right caudal

The allocation of the different test item dilutions to the sites (D-K) on the four 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 50% and 3%, 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 purified 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) Purified water
3) 1 :1 (w/w) mixture of purified 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 scapulat arca (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2x 4 cm patch of filter paper was saturated with the test item (50% in purified water) and placed over the injection sites of the test animals. The volume of test item preparation 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 imperuious 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 purified water only, also 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 3 % (applied to the left flank) and the vehicle only (purified 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 Íor 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 chatlenge application were compared with the results seen in control animals. An allergic reaction was defined by visible reddening of the challenge site. lf 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. lf the dermal reactions of test animals following the challenge were not clearly ditferent from the reactions seen in the control group animals, the results for the tesf 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 peisistent than the reactions observed in the controlanimals.
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
No necropsies were performed in the animals of the epidermal pretest ll, control and test group sacrificed at termination of the observation period nor in the animals of the intradermal and epidermal pretest I sacrificed on test day 1 of the main study.
The animals were sacrificed by intraperitoneal injection of NARCOREN 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):

yes

Remarks:

The sensitivity and reliability of the experimental technique employed was assessed by use of ALPHA-HEXYLCINNAMALDEHYDE which is recommended by the OECD 406 Guidelines and is known to have moderate skin sensitization propedies in the guinea pig strain.

Results and discussion

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 ALPHA-HEXYLCINNAMALDEHYDE was selected as a positive control. This was performed in accordance with the recommendation of the OECD TG 406, adopted bythe 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 Füllinsdorf / Switzerland).
The intradermal induction of sensitization in the test group was performed in the nuchal region with a 10 % dilution of the test item in PEG 300 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 10 % in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FOA/physiological saline and epidermally induced with PEG 300 under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 0.1 % in PEG 300 and PEG 300 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 test animals (at the 24-hour reading) and 6 out of 10 animals (at the 48-hour reading) showed discreteipatchy erythema after the challenge treatment with ALPHA-HEXYLCINNAMALDEHYDE at 0.1% (w/w) in PEG 300. No skin etfect 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, ALPHA-HEXYLCINNAMALDEHYDE have to be classified and labelled as a skin sensitizer.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Induction with purified water only. Challenge: purified water only and test item at 3%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were observed

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Induction with purified water only. Challenge: purified water only and test item at 3%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions were observed

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induction with test item (10% in water). Challenge: purified water only and test item at 3%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

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

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induction with test item (10% in water). Challenge: purified water only and test item at 3%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

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

Any other information on results incl. tables

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

Animals nos. 693 and 698 of the test group showed a loss of body weight (2.2 to 5.4 %) during the acclimatization period and animal no.688 of the control group did not gain body weight during the same phase. However, 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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on criteria defined in Regulation (EC) No 1272/2008, Red Rwa 4565 is considered to be "not sensitzing" to guinea pigs and is not classified according to CLP criteria.

Executive summary:

ln order to assess the cutaneous allergenic potential of Red RWa 4565, 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 96/54IEEC, 8.6.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 10 % dilution of the test item in purified 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 50 % in purified water one week after the intradermal induction. The animals of the control group were intradermally induced with purified water and FCA/physiological saline and epidermally induced with purified water under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item al3 o/o in purified water and purified 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.

None of the control and test animals showed skin reactions after the challenge treatment with Red RWa 4565 at 3% (w/w) in purified water.