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Administrative data

Description of key information

Skin sensitisation (OECD 406): not skin sensitising

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:
17 Feb - 02 Apr 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
Buehler test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in-vivo testing was set into force.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 8 - 12 weeks
- Weight at study initiation: 338 - 426 g
- Housing: singly or in pairs in solid-floor polypropylene cages, bedding woodflakes
- Diet: Guinea Pig FD1 Diet (Special Services Limited, Witham, UK) ad libitum
- Water: mains tap water, ad libitum, analysis was performed
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 0 - 14
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphtalate 1:1
Concentration / amount:
50% (v/v) and 100%
Day(s)/duration:
Day 28
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (controls), 20 (test groups)
Details on study design:
RANGE FINDING TESTS:
The concentrations of test material to be used at each stage of the main study were determined by sighting tests in which groups of guinea pigs were treated with various concentrations of test material. 4 animals were used: 2 animals for the topical induction and 2 for the topical challenge.
For the topical induction 2 animals were treated with 4 concentrations of the test item (10%, 25%, 50% and 100%). Applications were made to the clipped flanks under occlusive conditions for 6 h. The highest concentration of the test item producing only minimal dermal irritation was selected for the topical induction stage of the main study.
For the topical challenge 2 animals were treated with 2 concentrations of the test item (50% and 100%). Previously, the animals had been treated identically to the control animals of the main study an Days 0, 7 and 14. Applications were made to the clipped flanks under occlusive conditions for 6 h. The highest concentration of the test item which produced no evidence of dermal irritation, and 50% of the maximum non-irritant concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: undiluted test substance
- Control group: a blank patch was applied
- Site: left flank
- Frequency of applications: every 7 days
- Duration: Days 0-14
- Concentrations: 100%, under occlusive conditions

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 28
- Exposure period: 6 h
- Test groups: test substance and test substance in ethanol/diethylphthalate 1:1
- Control group: test substance and test substance in ethanol/diethylphthalate 1:1
- Site: right flank (100% test substance) and a separate skin site on the right flank (50% test substance)
- Concentrations: 50% and 100%
- Evaluation (hr after challenge): 24 and 48 h after patch removal
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole, topical induction and challenge: 50% in acetone/Peg 400 (70:30)
Positive control results:
A reliability test with 2-mercaptobenzothiazole (50% in acetone/Peg 400 (70:30)) was performed not more than 6 months previously (Feb 1999) using the same animal supplier. The incidence of sensitisation was 55% (11 out of 20 animals).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; Challenge: 50% and 100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 100%; challenge: 50% and 100%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; challenge: 50% and 100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100%; challenge: 50% and 100%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
other: not specified
Hours after challenge:
24
Group:
positive control
Dose level:
50% in induction and challenge
No. with + reactions:
11
Total no. in group:
20

Bodyweight gains of animals in the test group, between Day 0 and Day 30, were comparable to those observed in the control group animals over the same period.

No skin reactions were noted at the topical induction sites of test or control group animals at the 1, 8 or 15-day observations.

No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the Buehler test the test substance revealed no sensitising properties.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of the test substance was determined by a Buehler (Key study) and a guinea pig maximisation test (Supporting study). In addition, one LLNA test performed in 1995 showing potential sensitising effects using test substance concentrations of 1, 3 and 10% in acetone is available, but was disregarded due to major methodological deficiencies. The study was conducted according to GLP and equivalent to OECD Guideline 429. However, male, not female, CBA mice were used within the study and acetone was used as a non-standard vehicle which is known to enhance transdermal permeation (Marwah et al., 2014). Further, skin reactions (irritation scores and ear thickness) at application site was not evaluated. In addition, EC3 of positive control was below acceptable range of 4.4 - 14.7 as given in OECD TG 429, Table 1 (2010), thus the assay was possibly too sensitive. In conclusion, this study was disregarded.

In a first sensitisation study, the test substance was investigated in a Buehler test according to OECD Guideline 406 and in compliance with GLP (1999). The concentrations of test material to be used at the main study were based on a range finding study.

In the main study, 20 animals were used to investigate the skin sensitising potential of the test substance. In addition, 10 animals treated with vehicle served as control. For the topical induction with the undiluted test substance, applications were made to the clipped flanks under occlusive conditions for 6 h on Day 0, 7 and 14. For the topical challenge on Day 28 undiluted and 50% solution test substance in ethanol/diethyl phthalate 1:1 of were apllied to the clipped flanks under occlusive conditions for 6 h.

The undiluted test substance revealed no skin reactions at the topical induction sites on Days 0, 7 and 14. No skin reactions were observed after challenge with undiluted test substance and a 50% solution of the test substance. Body weight gains of animals in the test group, between Day 0 and Day 30, were comparable to the control group.

Based on the results of this Buehler test, the test substance was not regarded as a skin sensitiser under the conditions of the test.

 

In a second sensitisation study, the test substance was investigated in a guinea pig maximisation test (GPMT) according to OECD Guideline 406 and in compliance with GLP (1989). A range finding study was performed to determine the appropriate dose level of the test substance following intradermal and epicutaneous administrations. For the intradermal administration test substance concentrations of 0.625, 1.25, 2.5 or 5% were injected intradermally ten times in two animals each. No skin reactions were observed up to the highest concentration tested. For the epicutaneous administration test substance concentrations of 25, 50 and 100% were applied each to the skin of two animals. Exposure to the undiluted test substance resulted in slight erythema. No skin reactions were observed at 25 and 50%. In order to find the highest non-irritant test substance concentration for the challenge application test substance concentrations of 12.5, 25 and 50% were applied epicutaneously to the skin of four animals each. Slight erythema (score 1) was observed as a result of the treatment with 50% test substance concentration. No skin reactions were observed at 12.5 and 25%. A concentration of 5% (w/w) in paraffin oil was selected for the intradermal induction. A 100% test concentration was chosen for the epicutaneous induction and a concentration of 25% (w/w) in diethyl phthalate was selected for the challenge application.

In the main study, 20 animals were used to investigate the skin sensitising potential of the test substance. In addition, 20 animals treated with vehicle served as control. In the first induction stage no skin reactions were observed following the intradermal injections of 0.05 mL test substance. Intradermal injections of 0.05 mL FCA mixed with test substance or vehicle elicited irritation. On Day 6, 10% SDS was massaged with a glass rod into the skin of the same scapular area in order to provoke a mild inflammatory reaction. The challenge with 0.1 mL of the 25% solution of the test substance in diethyl phthalate on Day 21 revealed no positive skin responses in the control and test group. The animals made normal body weight changes over the duration of the study and exhibited normal appearance and behaviour.

Based on the results of this GPMT, the test substance was not regarded as a skin sensitiser under the conditions of the test.

Further data are available confirming the absense of the skin sensitising potential of the test substance. In human repeated insult patch tests (HRIPT), no reactions considered indicative of skin sensitisation were observed (Api et al., 2015).

 

References:

Api A. M.et al. (2015), RIFM fragrance ingredient safety assessment, allyl (cyclohexyloxy)acetate, CAS registry number 68901-15-5, Food and Chemical Toxicology, doi: 10.1016/j.fct.2015.03.016

Harneet Marwah, Tarun Garg, Amit K. Goyal & Goutam Rath (2016), Permeation enhancer strategies in transdermal drug delivery, Drug Delivery, 23:2, 564-578, DOI: 10.3109/10717544.2014.935532


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.