Trisodium hydrogen [2-[[α-[[3-[[4-chloro-6-[ethyl[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulphophenyl]azo]benzyl]azo]-4-sulphobenzoato(6-)]cuprate(4-)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation, other

Remarks:

in vivo (non-LLNA). Adequate data from an in vivo study are available

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From March 15th to April 08th, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines. Justification for Read Across is detailed in the endpoint summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 12th May 1981

Principles of method if other than guideline:

The test item was evaluated for potential skin sensitizing properties in a maximisation test as described by Magnusson and Kilgann.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: Hsd/Win:DH (previous designation: Bor=DHPW).
- Source: guinea pigs were bred by Harlan Winkelmann GmbH, Borchen/Paderborn
- Age at study initiation: about 5 to 7 weeks.
- Weight at study initiation: at the start of the experiment the mean initial weight of the animals was 331 g (298 to 360 g).
- Housing: during the acclimatization period the animals were housed conventionally in Type IV Makrolon cages (5 animals per cage). During the study period 2 or 3 animals were kept in one cage. The cages were exchanged for cages with clean bedding three times a week.
- Bedding: the bedding consisted of low-dust wood granules. The wood granules were randomly analysed for contaminants and the records are on file at testing laboratory. The analytic results provided no evidence of an effect on the study objective.
- Animgl Quarters: all animals of this study were placed in the same animal room. For reasons of capacity, guinea pigs from other toxicological studies were housed temporarily in that room. Confusion of animal numbers was avoided by adequate spatial separation, cage labelling and appropriate scheduling of operations.
- Cleaning, Disinfection: the animal room was swept daily with a broom and thoroughly cleaned with water once a week. At least once a month the animal room was disinfected with Zephirol 10 % (10 g benzalkonium chloride per 100 g; application dilution: 2 %, corresponding to 20 ml to 1 l water). Contamination of the feed and contact of the experimental animals with the disinfectant were avoided. No pest control was carried out in the animal rooms.
- Diet: the diet consisted of Altromin 3020 "Haltungsdiaet fuer Meerschweinchen" (maintenance diet for guinea pigs) manufactured by Altromin GmbH, Lage. Available ad libitum.
- Water: tap water, ad libitum. The tap water was of drinking quality.
- Acclimation period: after their arrival, the animals for this study were acclimatized to housing conditions for at least 7 days immediately prior to the start of the treatment; their health was monitored.
- Health/suitability check: the health status of the breed is routinely spot-checked for the most important specific infective agents and the results are on file. The sensitivity of this guinea pig strain for sensitization tests is verified and checked at regular intervals.
- Health status: only healthy animals, free of clinical signs, were used for this study. Neither before their delivery nor during the acclimatization or study period were the animals vaccinated or treated with anti-infectives. The females were nulliparous and not gravid.

ENVIRONMENTAL CONDITIONS
- Temperatur: 21 ± 1.5 °C (going upward at an outdoor temperature of more than 24 °C).
- Humidity: 40 - 70 %
- Air changes: about 12 - 15 times per hour.
- Photoperiod: 12 hours light/dark cycle, artificial illumination from 6 a.m. to 6 p.m.
There were occasional deviations from these standards, e.g. when the animal room was being cleaned. They had no noticeable effect on the study outcome.

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Concentration / amount:

Intradermal induction 5 % (i.e. 20 mg test compound/animal)
Topical induction: 50 % (i.e. 250 mg test compound/animal)
Challenge: 50 % (i.e. 250 mg test compound/animal) and 25 % (i.e. 125 mg test compound/animal)

Route:

epicutaneous, semiocclusive

Vehicle:

physiological saline

Concentration / amount:

Intradermal induction 5 % (i.e. 20 mg test compound/animal)
Topical induction: 50 % (i.e. 250 mg test compound/animal)
Challenge: 50 % (i.e. 250 mg test compound/animal) and 25 % (i.e. 125 mg test compound/animal)

No. of animals per dose:

Three groups of experimental animals were randomly established for the main study: one test substance group consisting of 20 experimental animals and two control groups consisting of 10 animals each.
The second control group was kept in reserve for a possible 2nd challenge, in which, if necessary, equivocal results of the 1st challenge could be checked or, if the results of the 1st challenge were positive, dose-dependencies could be investigated.

Details on study design:

TEST ITEM FORMULATION
Immediately prior to each application the test substance was formulated in sterile physiological saline solution as a suspension (w/v). Physiological saline solution corresponds best to the exposure conditions on the perspiring human skin.
During application the formulations were continuously mixed on a magnetic stirrer.

DOSE SELECTION
Dose selection for the induction and challenge application were based on the results of range-finding tests.

RANGE FINDING TESTS
Intradermal induction was carried out with a 5 % test compound formulation, topical induction with 50 %; theses concentrations had produced no primary skin irritant effects in the range-finding test.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction
One day prior to application the skin on the dorsum and the flanks was shorn. Starting behind the nucha, three intradermal injections were administered on each side of and parallel to the spinal column.
The application volume per injection site was 0.1 ml.
The animals of the three groups were treated as follows:
- Test substance group
1. injection site pair (cranial): Freund's complete adjuvant (Difco Lab.) 1:1 diluted with physiological saline solution (sterile).
2. Injection site pair (medial): test item 5%, formulated with physiological saline solution (sterile).
3. Injection site pair (caudal): test item 5%, formulated in physiological saline solution and Freund‘s complete adjuvant in equal proportions.
- Control groups
The animals of the control groups were treated like the animals of the test suhstance group, the formulation for the injection site pairs 2 and 3, however, contained no test compound but a corresponding amount of sterile physiological saline solution.

Topical induction
One week after intradermal induction topical induction took place.
On the eve, the areas to be treated were shorn, and a 10 % preparation of sodium lauryl sulphate in Vaseline was spread on these areas. Hypoallergenic patches (2 x 4 cm) were applied between or on the injection sites; they were covered with aluminium foil and kept in place on the skin with Fermoflex adhesive tape.
The hypoallergenic patches were treated as follows:
- Test substance group: 0.5 ml test item 50 %
- Control group; 0.5 ml physiological saline solution (sterile)
At the end of the 48-hour exposure period the substance residues were removed with tap water.

B. CHALLENGE EXPOSURE
The topical challenge took place 3 weeks after intradermal induction. The test compound concentration for challenge had been determined in a range-finding test on 5 guinea pigs treated like control animals during inductions.
One day prior to the treatment the animals were shorn on the dorsum and the flanks. For challenge a hypoallergenic patch saturated with 50 % and 25 % test compound formulation was placed on the left flank of the animals of the test substance group and the 1st control group. The patch was fixed for 24 hours on the skin with Fermoflex adhesive tape. The patches were applied in both concentrations cranially or caudally - alternately from animal to animal. On the right flank two control patches were fixed, saturated only with sterile physiological saline solution.
The application volume was 0.5 ml in each case.
At the end of the exposure period, substance residues were removed with tap water. 21 hours later the skin of the animals was depilated in the region of the treated areas. Pilca cream (depilation cream for humans, containing salts of the thioglycolic acid) was used for that purpose.

EVALUATION OF SKIN REACTIONS
The skin reactions were evaluated 48 and 72 hours after the start of application for challenge or the range-finding tests for topical induction and challenge. The following criteria were used.
Redness and escharosis:
0 = no reaction
1 = very slight redness (barely noticeable)
2 = well visible redness
3 = moderate to severe redness
4 = severe redness to slight escharosis (skin defects in depth)

Edema formation:
0 = no reaction
1 = very slight edema (barely noticeable)
2 = slight edema (edge of the area is well defined by marked eversion)
3 = moderate edema (eversion of area approx. 1 mm)
4 = severe edema (eversion more than 1 mm, spread out beyond the treated area)

Possible skin reactions which are not covered by these criteria are recorded in descriptive form.

ASSESSMENT OF RESULTS
To assess if and to what extent sensitization occurred, the number of animals of the test substance group and the control group with a skin irritant reaction and the severity of the reactions were established and a comparison was made.
The criterion for sensitization was whether the occurrence of skin irritant reactions was more frequent and severe for the animals of the test substance group than for the control group.

GENERAL EXAMINATIONS
During the entire study period the animals were observed for clinical signs at least once a day.
Body weights were determined prior to study initiation and on day 24, which was the last day of evaluation for the challenge exposure.

Challenge controls:

The reliability of the maximization test methods was checked in male guinea pigs using 2-mercaptobenzothiazole formulated in physiological NaCl. The following concentrations were used:
Intradermal induction was carried out with 2.5 %, topical induction with 40 % test compound formulation.
After the 1st challenge with 40 % and 12 % test compound formulation, 80 % and 55 %, respectively, of the test substance animals reacted with skin changes. After the 2nd challenge with 3 % and 1 % test compound formulation, 15 % each of the test substance animals showed skin findings. There were no skin reddenings in the control group after any challenge.

In a more recent test with 2-mercaptobenzothiazole and the same induction concentrations, 80 % and 65 % of the test substance animals reacted to the 40 % and 25 % test compound formulation, respectively, after the let challenge. After the 2nd challenge, 80 % and 75 % of the test substance animals responded to the 12 % and 6 % test compound, respectively, without any skin reddenings occurring in the control groups.
Thus, the sensitivity and reliability of the maximization test methods are verified by both studies.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole formulated in physiological NaCl

Remarks on result:

no indication of skin sensitisation

Appearance and behaviour of the animals treated with the test substance corresponded to those of the control animals.

From day 1 to day 13 the whole body of all test substance animals was discoloured in the colour of the test material.

On day 9 animals No. 13, 14, 15, 21 and 33 showed sporadic incrustations on the area treated during the 2^nd induction. On day 10 animals No. 11 and 35 and, on day 11, animal No. 32 also exhibited an incrustation on the area treated during the 2^nd induction. These incrustations had healed up in animals No. 11 and 13 on day 13, in animals No. 15, 33 and 35 on day 14 and in animals No. 14, 21 and 32 on day 16.

No deaths occurred.

Body weight development of the animals in the treatment group corresponded to that of the animals in the control group.

Incidences of skin reddenings after challenge

	Test item group (20 animals)					1^stcontrol group (10 animals)
	Test item patch			Control patch		Test item patch			Control patch
Hours	48	72	Total	48	72	48	72	Total	48	72
Challenge 25 %	0	1	1	0	0	2	1	2	0	0
Challenge 50 %	1	2	2	0	0	1	0	1	0	0

After challenge one animal (5 %) and two animals (10 %) of the test substance group reacted with skin reddening to the 50 % and 25 % test compound formulation, respectively. In the control group two animals (20 %) and one animal (10 %), respectively, showed skin reactions.

Interpretation of results:

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

Conclusions:

Under the conditions of the maximization test, the test compound has no skin sensitizing potential.

Executive summary:

The test substance was evaluated for potential skin sensitizing properties in a maximisation test as described by Magnusson and Kligmann. The test was performed on female guinea pigs. The following test compound concentrations were used in this test: intradermal induction 5 %; topical induction 50 %; challenge 50 % and 25 %.

The test compound was formulated in physiological saline solution (sterile) as a suspension. After challenge one animal (5 %) and two animals (10 %) of the test substance group reacted with skin reddening to the 50 % and 25 % test compound formulation, respectively. In the control group two animals (20 %) and one animal (10 %), respectively, showed skin reactions.

Conclusion

Under the conditions of the maximization test, the test compound has no skin sensitizing potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No experimental, nor literature data are available about the skin sensitisation of Reactive Blue 221 (RBl221), thus the available data on the Similar Substance 02, i.e. Reactive Blue FC 75311 (RBlFC750311) (EC: 419-480-1), have been considered.

The analogous substance was evaluated for potential skin sensitizing properties in a maximisation test as described by Magnusson and Kligmann. The test was performed on female guinea pigs. The following test compound concentrations were used in this test: intradermal induction 5 %; topical induction 50 %; challenge 50 % and 25 %.

The test compound was formulated in physiological saline solution (sterile) as a suspension. After challenge one animal (5 %) and two animals (10 %) of the test substance group reacted with skin reddening to the 50 % and 25 % test compound formulation, respectively. In the control group two animals (20 %) and one animal (10 %), respectively, showed skin reactions.

SIMILAR SUBSTANCE 02 - Reactive Blue FC 75311 (EC: 419-480-1)

The analogous substance RBlFC75311 (chemical structures in attachment) is the reaction mass of tetrasodium 2 -((1 -(3 -(6 -fluoro-(4 -((N-(2 -(2 -sulfatoethanesulfonyl)ethyl)-N-phenyl)amino)-1,3,5 -triazin-2 -ylamino)-2 -hydroxy-5 -sulfonatophenylazo)-1 -phenylmethyl)azo)-4 -sulfonatobenzoate cuprate (4 -) (main component 1) and trisodium 2 -((1 -(3 -((4 -(N-(2 -ethenesulfonylethyl)-N-phenyl)amino)-6 -fluoro-1,3,5 -triazin-2 -ylamino)-2 -hydroxy-5 -sulfonatophenylazo)-1 -phenylmethyl)azo)-4 -sulfonatobenzoate cuprate (4 -) (main component 2).

The main component 2 of RBlFC75311 shares the same structure of RBl221, except for the presence of the fluorine atom instead than the chlorine one, on the triazine ring; this difference is expected to not impact the potential for skin sensitisation (chlorine and fluorine are both halogens). The main component 1 represents a possible chemical precursor or transformation product of the main component 2, where the terminal sulphonate moiety has been lost, in the second case. RBl221 is expected to undergo to the same possible bond breaking in water phase, thus over a period of 48 hours (i.e. the experimental period) RBl221 is expected to be present as RBl221 as such and as transformed RBl21 (involving, as in the case of RBlFC75311, the lost of the sulphonate moiety).

The starting material in manufacture process should be the same (exception for the fluorine-chlorine difference) and the impurities profile is very similar: 60 - 100 % dye and the remaining composition including water (0 - 10 %), inorganic salts (e.g. sodium chloride/sulphate, etc, 0 -20 %) and a sum of by products and organic impurities in low concentrations (0 - 10 %, each impurity under 5 %).

RBlFC75311 is very water soluble (220 g/l at 20 °C and pH 6.8), like RBl221 (196.41 g/l at 20 °C and pH 4.18). Both substances are characterized by a very low affinity with the n-octanol phase, respect of the water one: the log Pow values of RBl221 and RBlFC75311 resulted to be negative (-2.5 and -2.7, respectively). Furthermore, possible reactivity/interaction with biological fluids and tissues are expected to be comparable.

Migrated from Short description of key information:
not sensitising

Justification for selection of skin sensitisation endpoint:
Study conducted on structural analogous according to internationally accepted testing guidelines.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin/respiratory sensitizer are reported into the second adaptation to technical progress*.

For Category 1, when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

Based on the response recorded in Guinea pig maximisation test, consequent to intradermal induction, a substance in classified as:

- skin sensitizer sub-category 1A, if the response is ≥ 30 % responding at ≤ 0.1 % intradermal inductiondose or if the response is ≥ 60 % responding at > 0.1 % to ≤ 1 % intradermalinduction dose

- skin sensitizer sub-category 1B, if the response is ≥ 30 % to < 60 % responding at > 0.1 to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermalinduction dose

In the case of the analogous substance Reactive Blue FC 75311 (EC: 419-480-1), the response resulted to be lower than 30 % responding at 5 % intradermal induction, thus also the substance under registration is expected to have no skin sensitizing potential.

In conclusion, Reactive Blue 221 does not meet the criteria to be classified as skin sensitizer, according to the CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures