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Description of key information

Reactive Red 66 was concluded to be a skin sensitiser in Guinea Pig Maximization Test.

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:
22 March 1993 to 24 June 1993
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 body weight of the animals was determined on acclimatization day 2 instead of acclimatization start due to technical reasons. The body weights at acclimatization start of some animals were minimal to slightly below the body weight range in protocol.
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation 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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 287380.26
- Expiration date of the lot/batch: November 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable; expiration date: November 1997
- Stability of the test substance in the solvent/vehicle: Unknown in bi-distilled water, FCA/physiological saline (50/50) and vaselinum album; therefore excluded from the Statement of Compliance
Species:
guinea pig
Strain:
Himalayan
Remarks:
Himalayan spotted
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 287.8 - 345.4 g
- Housing: Individually in Makrolon type-3 cages (size: 22 x 37 x 15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 342, Batches no. 74/92 (on the first acclimatization day) and 75/93 (from acclimatization day 2 to study termination) 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/1) via the drinking water.
- Acclimation period: One week for the control and test group under test conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + / - 3 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 to 15 air changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h artificiaî fluorescent light (approx. 100 lux) /12 h dark, music during the light period

IN-LIFE DATES: From: 22 March, 1993 To: 23 April, 1993
Route:
intradermal
Vehicle:
water
Remarks:
bi-distilled water
Concentration / amount:
5 %
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
other: vaselinum album
Concentration / amount:
25 %
Day(s)/duration:
Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline album
Concentration / amount:
25 % and 15 %
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
Details on study design:
RANGE FINDING TESTS:
A. INDUCTION EXPOSURE
- No. of exposures: 2 animals/dose
- Exposure period: 24 hrs
- Test groups: 3
- Control group: 1
- Site: clipped flank
- Frequency of applications: 1
- Duration: 8 days
- Concentrations: 5 %, 3 % and 1 %

B. CHALLENGE EXPOSURE
- No. of exposures: 1 animal /dose
- Day(s) of challenge: 2 weeks
- Exposure period: 24 hrs
- Test groups: 1
- Control group: 1
- Site: posterior left flank
- Concentrations: 25 %, 15 %, 10 %, 5 %
- Evaluation (hr after challenge): 24 hrs and 48 hrs
Challenge controls:
No positive reactions were evident after the challenge application, neither when treated with the vehicle (vaselinum album) alone nor when treated with test article concentrations of 25 % and 15 % in vaselinum album.
Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
12
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 %
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 %
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Remarks:
2-Mercaptobenzothiazol with 50 % positive (5/10)
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Remarks:
2-Mercaptobenzothiazol with 50 % positive (5/10)

Main Study

SKIN EFFECTS AFTER THE EPIDERMAL INDUCTION:

CONTROL GROUP: No positive skin reactions were observed in the animals treated with vaselinum album only.

TEST GROUP: Because of the violet discoloration of the application site due to the staining nature of the test article erythema reactions could not be assessed. No oedema reactions were evident.

SKIN EFFECTS AFTER THE CHALLENGE:

CONTROL GROUP: No positive reactions were evident after the challenge application, neither when treated with the vehicle (vaselinum album) alone nor when treated with test article concentrations of 25 % and 15 % in vaselinum album.

TEST GROUP: After the challenge application 12 animals of the test group showed an erythema reaction (2 animals with grade 1, 8 animals with grade 2 and 2 animals with grade 3) at the 24-hour reading and 14 animals (erythema in 4 animals with grade 1, 8 animals with grade 2, and 2 animals with grade 3) at the 48-hour reading when treated with the test article concentration of 25 %. At the highest non-irritant concentration of 15 % erythema reactions were observed in 11 animals (all 4 animals with grade 1 and 2, 3 animals with grade 3) at the 24-hour reading and in 13 animals (6 animals with grade 1, 4 animals with grade 2, and 3 animals with grade 3) at the 48-hour reading. No skin reactions were observed with vaselinum album alone applied at the first and second challenge procedure. Because the skin of the application sites was violet-discolored when treated with the test article dilution, depilation was carried out after removal of the dressing at the challenge. This enabled to observe clearly any possible erythema effect.

VIABILITY/MORTALITY: No deaths occurred during the entire study period.

CLINICAL SIGNS, LOCAL:

CONTROL GROUP: At the intracutaneous induction areas 1, 2 and 3 erythema and oedema was observed between day 2 and 4. At the intracutaneous injection sites 1 and 3 necroses were evident between day 5 and 8 and encrustations between day 10 and 14. Exfoliation of encrustation was noted at the injection sites 1 and 3 between day 15 and 26 (termination of the study). After the challenge application with the test article dilution, the treated skin was discolored from day 23 to 26.

TEST GROUP: At the intracutaneous induction areas 1 and 3 erythema and oedema respectively oedema and discoloration were observed between day 2 and 4, at the induction site 2 oedema between day 2 and 4, and discoloration from day 2 to 8. At the intracutaneous injection sites 1 and 3 necroses were evident between day 5 and 8 and at induction site 2 from day 6 to 8. Encrustations were noted at the intracutaneous injection sites 1, 2 and 3 between day 10 and 14. Exfoliation of encrustation was observed at the injection sites 1 and 3 between day 15 and 26 (termination of the study), at induction site 2 from day 15 to 19. The epidermal induction site was discolored from day 10 to 17. After the challenge application with the test article dilution, the treated skin was discolored from day 23 to 26. On day 9 of test no observation could be performed because the animals were bandaged semi-occlusively.

CLINICAL SIGNS, SYSTEMIC No clinical signs of systemic toxicity of the test article were observed in the control and test animals.

BODY WEIGHTS: Animal no. 135 of the e.c. pretest group slightly lost weight (-7.5 g) from acclimatization day 2 to 7. The body weight of the other animals was not affected adversely during the study.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
FAT 92354/A was found to be a skin sensitiser in the current Guinea Pig Maximization Test.
Executive summary:

The skin sensitisation study was carried out to assess the allergenic potential of FAT 92354/A when administered to the skin of guinea pigs by following: EU B.6.and OECD 406 guidelines. Ten females were used as control group and 20 females were used as test group. Due to unequivocal results obtained during the first challenge, no second challenge was performed. The highest non-irritating test article concentration used for the first challenge application was 15 %. Additionally, the 25 % concentration was used for the challenge application, because only one animal showed erythema reaction at the epidermal pretest at that concentration. There were no mortality and no adverse effects on body weight or systemic clinical signs were seen.


 


The skin effects observed after the challenge are as


CONTROL GROUP:


No positive reactions were evident after the challenge application, neither when treated with the vehicle (vaselinum album) alone nor when treated with test article concentrations of 25 % and 15 % in vaselinum album.


TEST GROUP:


After the challenge application 12 animals of the test group showed an erythema reaction (2 animals with grade 1, 8 animals with grade 2 and 2 animals with grade 3) at the 24-hour reading and 14 animals (erythema in 4 animals with grade 1, 8 animals with grade 2, and 2 animals with grade 3) at the 48-hour reading when treated with the test article concentration of 25 %.


At the highest non-irritant concentration of 15 % erythema reactions were observed in 11 animals (each 4 animals with grade 1 and 2, 3 animals with grade 3) at the 24-hour reading and in 13 animals (6 animals with grade 1, 4 animals with grade 2, and 3 animals with grade 3) at the 48-hour reading.


No skin reactions were observed with vaselinum album alone applied at the first and second challenge procedure. Because the skin of the application sites was violet-discoloured when treated with the test article dilution, depilation was carried out after removal of the dressing at the challenge. This enabled to observe clearly any possible erythema effect.


 


Based on the study results, FAT 92354/A is concluded to be a skin sensitiser in this Guinea Pig Maximization Test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitisation study was carried out to assess the allergenic potential of FAT 92354/A when administered to the skin of guinea pigs by following: EU B.6.and OECD 406 guidelines. Based on the study results, FAT 92354/A is concluded to be a skin sensitiser in Guinea Pig Maximization Test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the findings in the skin sensitisation study, the Reactive Red 066 considered to be classified as Skin Sensitiser (cat 1b) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.