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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 04. Jun. 1996 to 05. Jul. 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1993. Modified Buehler Method
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Constituent 1
Reference substance name:
-
EC Number:
424-510-1
EC Name:
-
Cas Number:
220150-59-4
Molecular formula:
not applicable for UVCB substance
IUPAC Name:
Reaction products of Phenol, 2,4-dinitro-, sulfurized, leuco derivatives and (3-chloro-2-hydroxypropyl)trimethylammonium chloride

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Ibm: GOHI; SPF-quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL. Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Füllinsdof, CH
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 5 to 7 weeks
- Weight at study initiation: 284 to 374 g
- Housing: individually in Makrolon type-3 cages with standard softwood bedding S 8/15, batch 231 ("Lignocell", Schill AG, 4132 Muttenz, CH)
- Diet: ad libitum; pelleted standard Nafag Ecosan 845 25W4, batch no. 32/96 guinea pig breeding/maintenance diet ("Nafag", Nähr- und Futtermittel AG, 9202 Gossau, CH); results of bacteriological, chemical and contaminant analyses included in report
- Water: ad libitum; community tap water from Itingen; once weekly supply of ascorbic acid (1 g/l) added; results of bacteriological, chemical and contaminant analyses included in report.
- Acclimation period: seven days for the control and test groups 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
- Indication of any skin lesions: only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3
- Humidity: 40 to 70 %
- Air changes: 10 to 15 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
50 %
Day(s)/duration:
6 hours, three times, separated by 1 week
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
50 %
Day(s)/duration:
6 hours, once
No. of animals per dose:
Induction screening test group: 4 female guinea pigs
Challenge screening test group: 4 female guinea pigs
Test group: 20 female guinea pigs
Control group: 10 female guinea pigs
Total number of animals used: 38 female guinea pigs
Details on study design:
INDUCTION SCREENING TEST:
- No. of exposures: 1
- Exposure period: 6 hours
- Animals: screening group 1: 4 female guinea pigs
- Site: fur was shaved with a fine clipper blade from the left and right posterior quadrant of the side and back of the animals
- Application: one application to each of four quadrants; allocation of different test dilutions was alternated in order to minimise site to site variation in responsiveness
- Concentrations: 50, 25, 15 and 10 % (w/w) test item in bi-distilled water
- Observations: erythema and oedema scores were taken after 24 and 48 hours; depilated 21 hours after end of treatment (VEET Cream, Reckitt & Colman AG, 4123 Allschwill, CH)
- Results: no oedema events; 2 animals demonstrated minore (±0) erythema after 24 hours which was reversible within 48 hours. 50 % test item concentration was selected as the most representative concentration to stimulate a state of immune hypersensitivity for the induction test.

INDUCTION EXPOSURE
- No. of exposures: 3 (study days 1, 8 and 15)
- Exposure period: 6 hours
- Test groups: 20 female guinea pigs received treatment
- Control group: 10 female guine pigs received no treatment
- Site: fur was clipped from the left posterior quadrant of the side and back of the animals
- Application: test item was applied at the cranial end of the site and covered with a patch
- Frequency of applications: weekly
- Concentration: 50 % (w/w) in bi-distilled water
- Evaluation: erythema and oedema were assessed 24 hours after patch removal without depilation

CHALLENGE SCREENING TEST:
- No. of exposures: 1
- Exposure period: 6 hours
- Animals: screening group 2: 4 female guinea pigs
- Site: fur was shaved with a fine clipper blade from the left and right posterior quadrant of the side and back of the animals
- Application: one application to each of four quadrants; allocation of different test dilutions was alternated in order to minimise site to site variation in responsiveness
- Concentrations: 50, 25, 15 and 10 % (w/w) test item in bi-distilled water
- Observations: erythema and oedema scores were taken after 24 and 48 hours; depilated 21 hours after end of treatment (VEET Cream, Reckitt & Colman AG, 4123 Allschwill, CH)
- Results: no oedema or erythema events observed; 50 % test item concentration was selected as the most representative concentration to stimulate a state of immune hypersensitivity for the challenge test.

CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: day 29
- Exposure period: 6 hours
- Test groups: 20 female guinea pigs received treatment
- Control group: 10 female guinea pigs received treatment
- Site: fur was clipped from the left posterior quadrant of the side and back of the animals
- Application: test item was applied at the caudal end of the site and covered with a patch
- Concentrations: 50 % (w/w) test item in bi-distilled water
- Evaluation: erythema and oedema were assessed 24 and 48 hours after end of treatment without re-depilation
Challenge controls:
Induction: received no treatment
Challenge: received same treatment as test animals
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (purity 85 %) in a concurrent reference study

Results and discussion

Positive control results:
58% of the animals of the test group were observed with positive reactions (grade of 0 and + are considered to be non-significant responses, whereas those of 1 and greater are considered to be significant) after challenge performed with the highest non-irritating concentration of ALPHA-HEXYLCINNAMALDEHYDE at 5% in polyethyleneglycol (PEG 400).
No reactions were observed in the control group treated in the same conditions during the challenge phase.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 % (w/w)
No. with + reactions:
0
Total no. in group:
19
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 % (w/w)
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:
50 % (w/w)
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
Dose level:
25 % in polyethyleneglycol
No. with + reactions:
11
Total no. in group:
19
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 % in polyethyleneglycol
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Mortality: one animal of the test group was found dead on test day 14. At necropsy, a dark red discolouration of the lungs was observed.

Clinical signs: no systemic symptoms were observed during the study

Body weights: one animal of the induction screening group and one animal of the challenge screening group lost weight during their treatment

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP criteria (EC 1272/2008)
Conclusions:
The substance was not found to be a skin sensitiser under test conditions.
Executive summary:

Skin sensitisation potential was evaluated in an experimental study according to the "Buehler Test" (Guinea Pig Sensitisation Testing by the Buehler Topical Closed Patch Technique by Ritz, H.L., and Buehler, E.V., 1979) as specified in the OECD Guideline 406 (1992) and the EU Method B.6 (1992).

An induction screening test was first performed (four female animals were administered 10, 15, 25 and 50 % (w/w) to four different quadrants of the back for 6 hours and were monitored for erythema and oedema 24 and 48 hours after exposure), which established a representative concentration for immune hypersensitivity stimulation of 50 % (w/w) test item in bi-distilled water. In the induction application, 20 female animals were administered 50 % (w/w) test item in bi-distilled water to the left, cranial, posterior quadrant of the back for 6 hours, three times spaced one week apart. Erythema and oedema were assessed after 24 hours.

A challenge screening test was first performed (four female animals were administered 10, 15, 25 and 50 % (w/w) to four different quadrants of the back for 6 hours and were monitored for erythema and oedema 24 and 48 hours after exposure), which established 50 % (w/w) test item as an appropriate challenge test concentration based on the highest non-irritating concentration. On test day 29, the test animals plus an additional group of control animals (10 females) were administered 50 % (w/w) test item in bi-distilled water for 6 hours, one time to the left, caudal, posterior quadrant of the back. Erythema and oedema were assessed 24 and 48 hours after patch removal.

One animal from the test group was found dead on day 14. Post-mortem macroscopic examination demonstrated strong red discolouration in the lungs of this animal. One induction screening animal and one challenge screening animal lost weight during their treatment. No skin reactions were observed among test or control animals after the challenge performed with test item at the highest non-irritating concentration 50 % in bi-distilled water.

Therefore, the substance has no skin sensitising potential.