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Skin sensitisation

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Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was performed before the validation of LLNA OECD method and is considered to be valid and acceptable for the assessment.
Species:
guinea pig
Strain:
other: Ibm:GOHI (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Fullinsdorf.
- Age at study initiation: 8 weeks.
- Weight at study initiation: 396 - 432 g.
- Housing: individually in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 342, Batch 61/90 guinea pig breeding/ maintenance diet (Klingentalmuhle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1g/l) via the drinking water.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 air change/hour.
- Photoperiod: 12 light cycle day.
Route:
intradermal and epicutaneous
Vehicle:
other: intraderma: physiological saline, epicutaneous: 25 % in vaselinum album.
Concentration / amount:
1st: induction 1 % intracutaneous.
2nd: induction 25 % occlusive epicutaneous.
3rd: challenge 25 % occlusive epicutaneous.
Route:
epicutaneous, semiocclusive
Vehicle:
other: intraderma: physiological saline, epicutaneous: 25 % in vaselinum album.
Concentration / amount:
1st: induction 1 % intracutaneous.
2nd: induction 25 % occlusive epicutaneous.
3rd: challenge 25 % occlusive epicutaneous.
No. of animals per dose:
Control group: 10
Test group: 20
Pre-test: 6
Details on study design:
RANGE FINDING TESTS
Intradermal injections (0.1 ml/site) were made into the clipped flank of 2 guinea pigs at 1, 3, and 5 %. The resulting dermal reactions were scored 24 hours later. Patches of filter paper (2 x 2 cm) were covered with a thin layer of test material in vaselinum album at 5, 10, 15 and 25 % and applied to the clipped and shaved flanks of each of 4 guinea pigs. The patches were covered with a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. The dressings were removed after 24 hours and the reactions were scored immediately and 24 and 48 hours later.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal: 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. Test animals were injected with Freund's complete adjuvant:physiological saline (50:50), 1 % test material (in physiological saline) or test material diluted to 1 % by emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline at the 3 sites. Control animals were injected with Freund's complete adjuvant:physiological saline (50:50), physiological saline, or a 50:50 mixture of Freund's complete adjuvant and physiological saline.

Epidermal: On day 7 of the test (approximately 24 hours prior to epidermal application), the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and pretreated with 10 % sodium-lauryl-sulfate (SLS) in petrolatum oil, because none of the concentrations given previously in the pretest (up to 25 %) caused irritation. The SLS was massaged into the skin with a glass rod without bandaging. The treatment provoked a mild inflammatory reaction. A day later, a 2 x 4 cm patch of filter paper was covered with a thin layer of the selected test material concentration (25 % in vaselinum album) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animals and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The control group was treated similarly with the omission of the test material. Reaction sites were assessed for erythema and edema immediately, and 24 and 48 hours after removal of the dressing.

B. CHALLENGE EXPOSURE
Challenge: Test and control animals were challenged 2 weeks after the epidermal induction and application. Hair was clipped from a 5 x 5 cm area on the left and right flank of each animal. Two patches (2 x 2 cm) of filter paper were covered with a thin layer of a non-irritant concentration of test material (25 % in vaselinum albumin) and with vaselinum album only, applied to the left and right flank using the same method as for the epidermal application. The dressings were removed approximately 18 hours later. The sites were assessed for erythema and edema immediately, and 24 and 48 hours after removal of the dressings. Control animals were treated similarly, omitting the test material. All animals were euthanized at the end of the test with an i.p. injection of pentobarbital (> 800 mg/kg).

OTHER:
An allergic reaction was defined by visible reddening of the challenge site. If the dermal reactions of test animals at challenge were more marked than controls, the animals were considered to have evidence of contact hypersensitivity. If they were not clearly different, they were considered to be "inconclusive". If they were identical to or less than the controls, the animals were considered to have no evidence of contact hypersensitivity.
 
Animals were observed daily for viability/ mortality. They were weighed at the beginning of acclimatisation, at day one, and at study termination.
Positive control substance(s):
yes
Remarks:
potassium dichromate
Positive control results:
For the induction period a 10% dilution of POTASSIUM-DICHROMATE in physiological saline and for the challenge procedure a 2.5% dilution of POTASSIUM-DICHROMATE in physiological saline was used. According to the results observed (8/10 positive) it is considered that the known allergen POTASSIUM-DICHROMATE possess a strong skin sensitizing potential in the guinea pig strain used.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2.5 %
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 2.5 %. No with. + reactions: 8.0. Total no. in groups: 10.0.

No positive reactions were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 25 % test article dilution.

 

Pre-test

During the pretest, all scores for erythema and edema were 2 (well defined) at all concentrations (with the exception one animal treated with 1 % that had a score of 1 [barely perceptible] for erythema). According to the findings observed, the concentration selected for the main study was 1 %. Epidermal application: All concentrations tested produced erythema scores of 1 (barely perceptible) immediately after exposure, with the exception that 5 and 10 % produced scores of 0 in one animal at this time. All other values were 0. Based on the findings, 25 % was selected for induction and challenge.

 

Main study

Induction

All ten control animals had erythema scores of 1 (barely perceptible) immediately after removal of the bandage. One had an edema score of 1 immediately after removal of the bandage. This same animal had an erythema score of 1, 24 hours after removal. All other control scores were 0. Nine out of twenty test animals had erythema scores of 1 (barely perceptible) immediately after removal of the bandage. One test animal had an erythema score of 2 at this time. Seven out of 20 test animals had edema scores of 1 immediately after removal of the bandage. Six and two test animals had erythema scores of 1, 24 and 48 hours after bandage removal (respectively). One animal had an edema score of 1 at 24 and 48 hours.

 

Challenge

The material was not sensitizing. All controls challenged with the vaselinum album had erythema scores of 1 immediately after bandage removal. All other control scores were 0. Nine out of 10 controls challenged with test material (25%) in vaselinum album were 1 immediately after bandage removal. All other scores were 0. Nineteen out of 20 animals induced with test material and challenged with vaselinum album had scores of 1 immediately after bandage removal. All other scores in this group were 0. All 20 animals induced and challenged with test material had scores of 1 immediately after bandage removal. All other scores in this group were 0.

Other

None of the animals died. No clinical signs were observed. Body weight gain of animals was not affected by treatment.

Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
not sensitising
Executive summary:

Method

The test substance skin sensatization potential was assessed in Albino Guinea Pigs. Maximization-Test, following the OECD guideline 406.

Results

No positive reactions were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 25 % test article dilution.

Conclusion

According to the CLP Regiulation, a substance is classified as skin sensitiser when an adjuvant type test method for skin sensitisation is used and the response of at least 30 % of the animals is considered as positive.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two studies have been conducted on Guinea Pig. The Key study (RCC, Research & Consulting Company AG. 1991) has been performed in GLP and the purity of the substance is known. For the supporting study the purity is unknown, but the two of them are confirming that the substance is not a skin sensitizer.

Furthermore a third study was performed on guinea pigs; unfortunately the original study reports is not more available, but there is only a summary reporting some toxicological test results on CAS 16090-02-1. The available information included in the toxicological test results summary indicate that the substance did not induced skin sensitization (Archroma GmbH, 1969).

REFERENCE

Archroma (1969). Toxikologische Untersuchung von Leukophor 6807. Unpublished data. Testing laboratory: Pharma-agroforschung. Owner company: Archroma GmbH. Report N. 22/69. Report date: 1969 -07 -02.

Migrated from Short description of key information:

Not skin sensitising (OECD 406) No effects in any animal

Justification for selection of skin sensitisation endpoint:

Study conducted according to internationally accepted testing guidelines and performed according to GLP. The sample tested is representative for the market batches.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Two studies were conducted on Guinea Pig. The Key study (Research & Consulting Company AG. 1991) was performed in GLP and the purity of the substance is known. No positive reactions were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 25 % test article dilution.

In the supporting study (Ciba Geigy Ltd., 1975) the composition of the lot tested is unknown. Nevertheless, no difference between the test and the control group was found after epidermal challenge and thus it was concluded that the substance is not sensitizing to skin.

Migrated from Short description of key information:

Not skin sensitising.

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 sensitizer, on the basis of results from test animals, are reported into the second adaptation to technical progress*: a substance in considered a skin sensitizer when:

- an adjuvant type test method for skin sensitisation is used and a response of at least 30 % of the animals is considered as positive;

- for a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive;

- a stimulation index of three or more is considered a positive response in the local lymph node assay.

No positive reactions were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 25 % test article dilution in both reported studies.

In conclusion, the available experimental data are adequate for classification and labelling and according to the CLP Regulation (EC 1272/2008), the test substance is not classified as skin sensitizing.

*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.