Octanoic acid, 6,8-dimercapto-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21.06.1999 - 01.08.1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

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:

The study was carried out at a time when LLNA-Studys was not yet available

Test material

Test material information

Constituent 1

Reference substance name:

6,8-disulfanyloctanoic acid

IUPAC Name:

6,8-disulfanyloctanoic acid

Constituent 2

Reference substance name:

6,8-disulfanyloctanoic acid

Cas Number:

462-20-4

Molecular formula:

C8H16O2S2

IUPAC Name:

6,8-disulfanyloctanoic acid

Constituent 3

Reference substance name:

6,8-disulfanyloctanoic acid

EC Number:

610-288-5

Cas Number:

462-20-4

Molecular formula:

C8H16O2S2

IUPAC Name:

6,8-disulfanyloctanoic acid

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Dihydrolipoic Acid
- Physical state: liquid
- Lot/batch No.: TC 3 KD 3
- Storage condition of test material: room temperature in the dark under nitrogen

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, Staffordshire, UK
- Age at study initiation: approx. 361 - 459 g
- Weight at study initiation: approx. 8 - 12 weeks
- Housing: Singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet: ad libitum Guinea Pig FD1 Diet, Special Diets Service Limited, Witham, Essex, UK
- Water: ad libitum tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): twelve hours continous light and twelve hours darkness

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

arachis oil

Concentration / amount:

5%

Day(s)/duration:

24/48 h

Adequacy of induction:

not specified

Challenge

No.:

#10

Route:

intradermal and epicutaneous

Vehicle:

arachis oil

Concentration / amount:

20

Day(s)/duration:

24/48 h

Adequacy of challenge:

not specified

No. of animals per dose:

20 animals for test / 10 animals for control

Details on study design:

RANGE FINDING TESTS:
The concentrations of the test material to be used at each stage of the main study were determined with "sighting tests" in which groups of guinea pigs were treated with various concentrations of the test material.

MAIN STUDY
A group of fifteen guinea pigs was used for the main study, ten test and five control. The bodyweight of each animal was recorded at the start and end of the study.
1) Induction
a) Induction of the test animals
Shortly before treatment on Day 0 the hair was removed from an area approx. 40 mm x 60 mm on the schoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line. The injections were:
- Freund's Complete Adjuvant plus distilled water in the ratio 1:1
- a 1 % w/v formulation of the test material in arachis oil in BO
- a 1 % w/v formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approx. 24 and 48 hours after intradermal injection the degree of erythema at the test material injection site was evaluated.
One week later (Day 7), the same area on the shoulder region used previously for intradermal injections was clippered again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No.4: approx. 40 mm x 20 mm), satured with the test material formulation (10 % v/v in arachis oil BP) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.
The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patch using the scale below. Any other reactions were also recorded.

b) Induction of the control animals
Intradermal injections were administered using an identical procedure to that used for the test animals, expect that the injections were:
- Freund's Complete Adjuvant plus distilled water in the ratio 1:1
- arachis oil BP
- 50 % w/v formulation of arachis oil BP in a 1:1 mixture of Freund's Complete Adjuvant/distilled water
The topical applications followed the same procedure as for the test animals expect that the vehicle alone was applied to the filter paper. Skin reactions were quantified as for the test animals.

2)Challenge
Shortly before treatment on Day 21, an area of approx. 50 mm x 70 mm on both flanks of each animals, was clipped free of hair with veterinary clippers.
A square filter patch (WHATMAN No. 4: approx. size 20 x 20 mm), satured with the test material formulation at the maximum non-irritant concentration (10 % v/v in arachis oil BP) was applied to the shorn right flnak of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 5 % v/v in arachis oil BP was similary applied to a skin site on the left shorn flank. The patches were occluded with an overlapping lenght of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully cut using blunt-tipped scissors, removed and discarded. The challange site were swabbed with cotton wool soaked in diethyl ether to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
Approx. 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified using the scale shown below. Any other reactions were also recorded.

3) Interpretation of results:
a) The percentage of the test animals that showed a more severe reaction at the test material challenge site than the most severe reaction seen in the control animals, was compared with the following scale:
Percentage of animals sensitised: Classification of sensitisation potential:
0 non-sensitiser
> 0 - 8 weak sensitiser
> 8 - 28 mild sentisiser
> 28 - 64 moderate sensitiser
> 64 - 80 strong sensitiser
> 80 extreme sensitiser
b) Evaluation of Erythema
0: No erythema
1: Discrete or patchy erythema
2: Moderate and confluent erythema
3: Intense erythema and swelling
Source: OECD Test Guideline 406, 1992 and Methode B.6 Skin Sensitisation of Comission Directive 96/54/EC.
c) Evaluation of Oedema
0: No oedema
1: Very slight oedema (barely perceptible)
2: Slight oedema (edges of area well-defined by definite raising)
3: Moderate oedema (raised approx. 1 mm)
4: Severe oedema (raised more than 1 mm extending beyond the area of exposure)
Source: Draize, J.H. (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures of Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC, p.31.

Positive control substance(s):

yes

Results and discussion

Positive control results:

no positive control group was used

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

none

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

not measured/tested

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

1) Skin Reactions Observed After Intradermal Induction:

Individual reactions observed at the test material intradermal injection sites of the test group animals and vehicle intradermal injection sites of the control gropu animals were recorded. Discrete or patchy to moderate and confluent erythema was noted at the intradermal induction sites of all test group animals at the 24 -hour obervation. Discrete or patchy erythema was noted at the intradermal induction sites of all test group animals at the 48 -hour observation. Discrete or pathy erythema was noted at the intradermal induction sites of all control group animals at the 24 and 48 -hour observations.

2) Skin Reactions Observed After Topical Induction

Individual skin reactions observed at the topical induction sites of the test and control group animals were recorded.

Discrete or patchy to moderate and confluent erythema with or without very slight oedema was noted at the topical induction sites of all test group animals at the 1 -hour observation. Moderate and confluent erythema with or without very slight to slight oedema was noted at the topical induction sites of all test group animals at the 24 -hour observation. Bleeding from the intradermal injection sites was noted in five test group animals at the 1 -hour observation and one test group animal at the 24 -hour observation. Other skin reations noted at the topical induction sites of the test group animals at the 24 -hour observation were bleeding wound caused by animal scratching, small superficial scattered scabs and dried blood.

No skin reactions were noted at the topical induction sites of control group animals at the 1 and 24 -hour observations.

3) Skin Reactions Observed After Topical Challenge

Individual skin reactions at the challenge sites of the test and control group animals are given in table 1.

a) 10 % v/v Arachis Oil BP

Positive skin response (moderate and confluent erythema with or without very slight oedema) were noted at the challenge sites of four test group animals at the 24 -hour observation and one test group animal at the 48 -hour observation. Discrete or patchy erythema was noted at the challenge sites of six test group animals at the 24 -hour observation and eight test group animals at the 48 -hour observation. Small superficial scattered scabs were noted at the challenge site of one test group animal at the 48 -hour observation.

Discrete or patchy erythema with or without very slight oedema was noted at the challenge sites of two group animals at the 24 and 48 -hour observations.

b) 5 % v/v in Arachis Oil BP

Positive skin reponses (moderate and confluent erythema with or without very slight oedema) were noted at the challenge sites of three test group animals at the 24 -hour observation. Discrete or patchy erythema were noted at the challenge sites of six test group animals at the 24 and 48 -hour observations.

Discrete or patchy erythema was noted at the challenge sites of two control group animals at the 24 -hour observation and one control group animal at the 48 -hour observation.

Table 1: Individual Skin Reactions in test Animals at Challenge (Challenge Concentrations: 10 % and 5 % v/v)

Animal Number	Skin Reactions (Hours After Removal of Dressing)
	24 Hours						48 Hours
	5 %			10 %			5 %			10 %
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
1	2	1	-	2	0	-	1	0	-	1	0	-
2	1	0	-	2	1	-	0	0	-	1	0	Ss
3	1	0	-	2	0	-	0	0	-	1	0	-
4	1	0	-	1	0	-	1	0	-	1	0	-
5	2	1	-	1	0	-	1	0	-	0	0	-
6	1	0	-	1	0	-	1	0	-	1	0	-
7	1	0	-	1	0	-	1	0	-	1	0	-
8	1	0	-	1	0	-	0	0	-	1	0	-
9	0	0	-	1	0	-	0	0	-	1	0	-
10	2	0	-	2	0	-	1	0	-	2	0	-
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	1	0	-	1	0	-	1	0	-	1	0	-
15	1	0	-	1	1	-	0	0	-	1	0	-

Er = erythema; Oe = oedema; - = no other reaction noted; Ss = small superficial scattered scabs

4) Bodyweight

Bodyweight gains of guinea pigs in the test group, between Day 0 and Day 24, were comparable to those observed in the control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test material, Dihydrolipoic Acid, produced a 40 % (4/10) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.

Executive summary:

In order to assess the contact sensitisation potential of Dihydrolipoic Acid in the albino guinea pig a study according to OECD 406 ("Skin Sensitisation") and 96/54/EC B.6 was performed.

Ten test and five control animals were used for the main study. Based on the results of the sighting tests, the concentrations of the test material for the induction and challenge phases were selected as follows:

Intradermal Induction: 1% w/v in arachis oil BP

Topical Induction: 10 % v/v in arachis oil BP

Topical Challenge: 10 % and 5 % v/v in arachis oil BP

The test material produced a 40 % (4/10) sensitisation rate and was classified as a moderate sensitiser to guinea pig skin.