Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun - 26 Jul 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Directive 92/96 EEC

Deviations:

no

GLP compliance:

yes

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was started before LLNA as first-choice method for in-vivo testing was set into force.

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

EC Number:

817-761-7

Cas Number:

765293-81-0

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with Phytosterol, Isooctadecan-1-ol, Hexadecan-1-ol, Octadecan-1-ol and Docosan-1-ol

Test material form:

other: paste

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, UK (sighting tests) and Harlan UK Limited, Bicester, UK (main study).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 326 – 548 g
- Housing: Animals were housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet: Certified guinea pig Diet (Code 5026) supplied by PMI Nutrition International (Nottingham, UK) and IPS Product Supplies Limited, Wellingborough, UK), ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 30 -70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

0.1 mL 5% (v/v)

Day(s)/duration:

Day 0

Adequacy of induction:

other: 5% (v/v) of the test material caused mild to moderate skin irritation and was well tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

undiluted test material

Day(s)/duration:

Day 7

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Concentration / amount:

undiluted test substance and 75% (v/v) test substance in arachis oil treated in two sites

Day(s)/duration:

Day 21

Adequacy of challenge:

other: highest non-irritant concentration and one lower concentration

No. of animals per dose:

10 (test group), 5 (controls)

Details on study design:

Selection of concentration for intradermal induction:
Intradermal injections (0.1 mL/injection site) were made on the clipped shoulder of one guinea pig using a concentration of 5% (v/v) in arachis oil. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 h and 7 days after injection. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.

Selection of concentration for topical induction:
Two guinea pigs (intradermally injected with Freund's Complete Adjuvant eight days earlier) were treated with the undiluted test substance and with test material concentrations of 75, 50 and 25% (v/v) in arachis oil. Applications were made under occlusive dressings for 48 h. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 h after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.

Selection of concentration for topical challenge:
The undiluted test material and test material concentrations of 75, 50 and 25% (v/v) in arachis oil were applied to two guinea pigs under occlusive dressings for 24 h. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 h after dressing removal. The highest non-irritant concentration of the test substance and one lower concentration were selected for the topical challenge stage of the main study.

Based on the results of the sighting tests, 5% (v/v) concentration of the test material was determinded for intradermal injection, the undiluted test substance (100%) was selected for topical induction, the undiluted test substance and 75% (v/v) of the test material (two sites) was used for topical challenge in the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections; 0.1 mL each):
Injection 1: Freund’s Complete Adjuvant (FCA) plus distilled water in the ratio 1:1
Injection 2: 5% (v/v) test material in arachis oil
Injection 3: 5% (v/v) test material in a 1:1 preparation of FCA plus distilled water
Epicutaneous: undiluted test substance
- Control group:
Intradermal (3 pairs of injections; 0.1 mL):
Injection 1: FCA plus distilled water in the ratio 1:1
Injection 2: arachis oil
Injection 3: arachis oil at 50% in 1:1 preparation of FCA plus distilled water
Epicutaneous: arachis oil
- Site: shoulder region (intradermal and epicutaneous)
- Frequency of applications: every 7 days
- Duration: Days 0-7
- Concentrations: intradermal: 5% (v/v), epicutaneous: undiluted test substance

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day of challenge: 21
- Exposure period: 24 h
- Test groups: undiluted test substance and 75% (v/v) test substance in arachis oil
- Control group: undiluted test substance and 75% (v/v) test substance in arachis oil
- Site: right flank (undiluted test substance) and left flank (75% (v/v) test substance in arachis oil)
- Concentrations: undiluted test substance and 75% (v/v) test substance in arachis oil
- Evaluation (hr after challenge): 24 and 48 hours after challenge dressing removal

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

yes

Remarks:

In the last reliability check (22 May - 15 Jun 2002) 2-mercaptobenzothiazole was used as positive control substance (intradermal: 5% in arachis oil, topical: 50% in acetone:PEG 400 (70:30); challenge: 25 and 50% in acetone:PEG 400 (70:30)).

Results and discussion

Positive control results:

The positive control 2-mercaptobenzothiazole (reliability check of May/June 2002) induced positive reactions ins 8/10 guinea pigs (80%) thus meeting the reliability criteria for the guinea pig maximisation test (≥ 30% positive response).

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

intradermal induction: 5% (v/v); challenge: 75% (v/v)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

intradermal induction: 5% (v/v); challenge: undiluted test substance (100%)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

intradermal induction: 5% (v/v); challenge: 75% (v/v)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

intradermal induction: 5% (v/v); challenge: undiluted test substance (100%)

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

intradermal induction: 0%; challenge: 75% (v/v)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

intradermal induction: 0%; challenge: undiluted test substance (100%)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 7

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

intradermal induction: 0%; challenge: 75% (v/v)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 8

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

intradermal induction: 0%; challenge: undiluted test substance (100%)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 9

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

intradermal induction: 5% in arachis oil, topical induction: 50% in acetone:PEG 400 (70:30); challenge: 25 and 50% in acetone:PEG 400 (70:30)

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Table 1 Individual skin reactions at challenge

Group	Animal Number	Skin reactions (Hours after removal of dressings)
		75%						100%
		24 hours			48 hours			24 hours			48 hours
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
Test	1	0	0	-	0	0	-	0	0	-	0	0	-
	2	0	0	-	0	0	-	0	0	-	0	0	-
	3	0	0	-	0	0	-	0	0	-	0	0	-
	4	0	0	-	0	0	-	0	0	-	0	0	-
	5	0	0	-	0	0	-	0	0	-	0	0	-
	6	0	0	-	0	0	-	0	0	-	0	0	-
	7	0	0	-	0	0	-	0	0	-	0	0	-
	8	0	0	-	0	0	-	0	0	-	0	0	-
	9	0	0	-	0	0	-	0	0	-	0	0	-
	10	0	0	-	0	0	-	0	0	-	0	0	-
Control	11	0	0	-	0	0	-	0	0	-	0	0	-
	12	0	0	-	0	0	-	0	0	-	0	0	-
	13	0	0	-	0	0	-	0	0	-	0	0	-
	14	0	0	-	0	0	-	0	0	-	0	0	-
	15	0	0	-	0	0	-	0	0	-	0	0	-

* Er = Erythema, Oe = Oedema, - = No other reactions noted

Table 2 Scales for evaluation of skin reactions – Magnusson and Kligman maximization method

EVALUATION OF ERYTHEMA#	VALUE
No erythema	0
Barely perceptible erythema	±
Discrete or patchy erythema	1
Moderate and confluent erythema	2
Intense erythema and swelling	3
EVALUATION OF OEDEMA#	VALUE
No oedema	0
Very slight oedema (barely perceptible)	1
Slight oedema (edges of area well – defined by definite raising)	2
Moderate oedema (raised approximately 1 millimetre)	3
Severe oedema (raised more than 1 millimetre extending beyond the area of exposure)	4

Moderate and confluent erythema was noted at the intradermal induction sites of test group animals, discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of control group animals.

Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of test group animals, and bleeding from the intradermal injection sites was noted in one test group animals. Discrete or patchy erythema and very slight oedema was noted at the topical induction sites of control group animals.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

The test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitiser to guinea pig skin under the conditions of the test.