Sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

Acid Blue 225 is a skin sensitiser.

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:

24 May 1994 to 08 Sep 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

The temperature was between 22-25°C in the animal room. The EN-Number was corrected according to the data sheets received from the sponsor..

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

The temperature was between 22-25°C in the animal room. The EN-Number was corrected according to the data sheets received from the sponsor..

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4100 (Skin Sensitisation)

Deviations:

yes

Remarks:

The temperature was between 22-25°C in the animal room. The EN-Number was corrected according to the data sheets received from the sponsor..

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Specific details on test material used for the study:

Identification: POLAR BLAU RLS (FAT 21'036/D)
Description: Solid
EN-Number: 384481.22
Purity / formulation: approx. 87% (a.i)
Stability of test article: Stable at storage conditions; expiration date: 04/99
Storage conditions: At room temperature
Safety precautions: Gloves, goggles and face mask were obligatory to ensure personnel health and safety.

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland
Number of animals per test: 38 females
Age at delivery/acclimatization start: 5-7 weeks
Body weight at delivery/acclimatization start: Test and control animals 348 - 440 g. Animals used for Irritation Screen 378 - 445 g
Identification: By unique cage number and corresponding individual number of animals and restrainer.
Randomization: Randomly selected at time of delivery.

Acclimatization: Seven days for the control and test group under test conditions after health examination. However contrary to the test group the control group remained untreated during the 3 induction weeks. One day for the animals used in the irritation screen for induction. Three weeks for the animals of the irritation screen for challenge. Only animals without any visual signs of illness were used for the study.

Conditions:
Standard Laboratory Conditions.
Air-conditióned with 10-15 air changes per hour and continuously monitored environment with a temperature between 22-25 degrees centigrade, a relative humidity between 54.5-82 %, 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark, music during the light period.

Accommodation:
Individually in Makrolon type-3 cages with autoclaved standard softwood bedding S 8/15, batch 231 ("Lignocel", Schill AG, CH-4132 Muttenz).

Diet:
Pelleted standard Kliba 342, Batch nos. 62/94 (at delivery of the animals to June 2, 1994) and 63/94 (from June 3, 1994, to termination of test) guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst) ad libitum.

Water:
Community tap-water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water.

Route:

epicutaneous, occlusive

Vehicle:

other: vaselinum album

Concentration / amount:

25 %

Day(s)/duration:

3 applications, one application per week

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: vaselinum album

Concentration / amount:

25 %

Day(s)/duration:

14 days after last induction exposure

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Challenge: 20 test animals and 10 control animals
Irritation Screen: 8 animals

Details on study design:

TREATMENT AND OBSERVATION METHODS:
Patching Method: The same patching method was used for induction, irritation screens, challenge and re-challenge. The animal’s fur was shaved on the day before exposure with a fine clipper blade. Closed patches were applied to the animals as follows: A 25 mm Hill Top Chamber was saturated with the test article dilution which was applied with a spatula. Animals were put in the restrainer and the designated patch was applied to the clipped surface as quickly as possible after the test substance had been applied. The patch appliance was occluded with a rubber dental dam. The rubber dental dam used for occlusion of the patches was of medium gauge 12 - 15 cm wide, depending on the animal size and the number of patches to be covered. The rubber dental dam was pulled snug on each side of the animal and secured with one or more large size clip on each side of the restrainer. The rubber dental dam was placed under the front and back metal restraining bands and had snug contact with the animal over the entire dorsal surface. The restrainers were adjusted to minimize movement of the animals during the exposure period. Six (6) hours (+ 15 minutes) later, the rubber dental dams and patches were removed, and the animals were taken away from the restrainers. After that the animals were returned to their individual cages.

GRADING METHOD:
The animals used for irritation screens and challenge were depilated eighteen to twenty-two (18 - 22) hours after patches had been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The depilatory 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. Erythema and edema were assessed as follows:

ERYTHEMA
0 = no erythema
+ = slight, patchy erythema (i.e barely perceptible or questionable reaction)
1 = slight confluent erythema (i.e., a slight but definite reaction at the patch site) or moderate, but patchy erythema (i.e., moderate erythema involving at least 50% or more of the area of the patch site)
2 = moderate confluent erythema
3 = severe erythema

OEDEMA (according to Draize)
0 = no edema
1 = very slight edema (barely perceptible)
2 = well-defined edema (edges of area well-defined by definite raising)
3 = moderate edema (raised approximately 1 millimeter)
4 = severe oedema (raised more than 1 millimeter and extending beyond the area of exposure)

The grading method used for irritation screens, induction and challenge was identical. This one was performed 24 hours (+ 2 hours) after removal of the patches for the irritation screens, induction and challenge and repeated 24 hours (+ 2 hours) later (48-hour grades) for irritation screen and challenge. The grading method used for re-challenge and challenge was identical.

NOTE:
(1) At challenge and re-challenge, control animals were graded before the test animals. Grading of all animals was done by positioning the animal under true light (Osram, 58-Watt, light quality TLD). Grading was done by two (2) technicians, who both agreed to the recorded score. Comparison of sites on other animals in the test or control groups was required to arrive at a final score.
(2) To monitor skin reactions during the induction phase, the skin was graded without depilation.

Positive control substance(s):

yes

Remarks:

0.5 % 1-chloro-2,4-dinitrobenzene in ethanol

Positive control results:

0.5 % 1-chloro-2,4-dinitrobenzene in ethanol positive control: In this study 100 % of the test animals showed significant positive skin reaction at the 24- and 48-hour reading. No reactions were observed in the control group.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

6

Total no. in group:

19

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

8

Total no. in group:

19

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.5 % in Ethanol (Induction concentration), 0.05% in Ethanol (Challenge concentration),

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.5 % in Ethanol (Induction concentration), 0.05% in Ethanol (Challenge concentration),

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

9

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

9

Remarks on result:

no indication of skin sensitisation

PRIMARY SENSITIZATION RESULTS (INCIDENCE TABLES)

CHALLENGE / Erythema Readings:

Induction Concentration (test group only): 25 % POLAR BLAU RLS (FAT 21036/D) in vaselinum album

Challenge Concentration (control & test group): 25 % POLAR BLAU RLS (FAT 21036/D) in vaselinum album

Grade of 0 and + are considered to be representative of insignificant responses, whereas those of 1 or greater are considered to be significant. In this study 42 % of the test animals were observed with significant response at either 24 or 48-hour challenge reading. The response of at least 15 % positive animals is considered positive "R43" following the commission 93/21/EEC, Commission Directive of 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 substance POLAR BLAU RLS (FAT 21036/D) tested in concentration of 25 % in vaselinum album is considered to be a sensitizer.

MORTALITY / VIABILITY / MACROSCOPIC FINDINGS: One animal (no. 652) of the control group was found dead on test day 28. At necropsy, a dark red discoloration of the uncollapsed lungs was observed as well as several dark red foci (D = 5mm). One animal (no. 678) of the test group was found dead on test day 1. At necropsy the lungs were not collapsed with several dark red foci (D = 4mm).

CLINICAL SIGNS (LOCAL/SYSTEMIC): Neither local nor systemic symptoms were observed during the study.

BODY WEIGHTS: Two animals of the control group and one animal of the test group lost weight during the acclimatization period. The lost of weight should be considered to be of incidental nature.

Table: Skin response after CHALLENGE / 25% POLAR BLAU RLS (FAT 21036/D) in vaselinum album

Animal #	Sex	Erythema (E) / Oedema (Oe) readings after removal of bandage
		24 hours		48 hours
		E	Oe	E	Oe
661	Female	1	0	1	0
662	Female	1	0	1	0
663	Female	±	0	1	0
664	Female	0	0	0	0
665	Female	0	0	±	0
666	Female	0	0	±	0
667	Female	1	0	1	0
668	Female	±	0	1	0
669	Female	±	0	±	0
670	Female	±	0	±	0
671	Female	0	0	0	0
672	Female	1	0	1	0
673	Female	2	0	2	0
674	Female	0	0	0	0
675	Female	±	0	0	0
676	Female	0	0	±	0
677	Female	0	0	0	0
678	Female	Was found dead on day 1
679	Female	0	0	0	0
680	Female	1	0	1	0

 ± = slight, patchy erythema (i.e.,barely perceptible or questionable reaction)

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

FAT 21036/D was found to be a skin sensitizer.

Executive summary:

A key study was performed to assess the possible allergenic potential of FAT 21036/D when administered to the skin of albino guinea pigs. For this purpose the "Buehler-Test" modified by Ritz, H. L. and Buehler, E. V. (1980) was used. Methodlogy was according to OECD test guideline 406 under GLP conditions. Ten females remained untreated and were used as control animals to be treated at challenge. Twenty test females were induced three times during induction and challenged with the test substance. Induction Concentration (test group only): 25 % FAT 21036/D in vaselinum album. Challenge Concentration (control & test group): 25 % FAT 21036/D in vaselinum album. Grade of 0 and + are considered to be representative of insignificant responses, whereas those of 1 or greater are considered to be significant. In this study 42% of the test animals were observed with significant response at either 24 or 48-hour challenge reading. No local or systemic symptoms were observed during the study. The response of at least 15 % positive animals is considered positive "R43" following the commission 93/21/EEC, Commission Directive of 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 substance FAT 21036/D tested in concentration of 25 % in vaselinum album is considered to be a sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A key study was performed to assess the possible allergenic potential of FAT 21036/D when administered to the skin of albino guinea pigs according to "Buehler-Test" modified by Ritz, H. L. and Buehler, E. V. (1980) was used. The test group animals received 3 exposures, one every week at 25 % concentration. Two weeks after the final induction exposure, the test and control animals were challenged with a 25 % concebtration. 42 % of the test animals were observed with significant response at either 24 or 48-hour challenge reading. Therefore, the test substance FAT 21036/D tested in concentration of 25 % in vaselinum album is considered to be a sensitizer.

FAT 21036/E was assessed for skin sensitisation potential in albino guinea pigs according to the maximization test protocol of B. Magnusson and A.M. Kligman (1969). This test was conducted in accordance with OECD test guideline 406 in a GLP certified laboratory. Based on the findings of the pretests, 5, 75 and 75 % were the concentrations used for intradermal induction, epidermal induction and epidermal challenege, respectively. 40 % of the animals of the test group were observed with positive skin reactions after the challenge. No skin reactions were observed in the control group. Therefore, FAT 21036/E is considered to be a sensitizer. In a supporting study, FAT 21036/C was also found to induce skin sensitisation when tested in guinea pigs using the maximisation as described by OECD Guideline 406. Thus, based on the outcomes of available studies, Acid Blue 225 induced sensitization in albino guinea pigs when tested as per Maximisation method as well as Buehler method.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Acid Blue 225 is to be classified as a sensitizer (cat 1B) according to the criteria of Regulation (EC) No. 1272/2008.