Potassium Lauroyl Wheat Amino Acids

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 07, 2001 to August 23, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Once the LLNA became an OECD method, the Buehler was already performed. Furthermore, the substance is irritating and for irritating substances a LLNA can be waived.

Test material

Test material information

Constituent 1

Reference substance name:

L-Glutamic acid, N-coco acyl derivs., monosodium salts

EC Number:

269-087-2

EC Name:

L-Glutamic acid, N-coco acyl derivs., monosodium salts

Cas Number:

68187-32-6

Molecular formula:

C5H8NaNO4· H2O

IUPAC Name:

L-Glutamic acid, N-coco acyl derivs., monosodium salts

Test material form:

not specified

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan spotted

Sex:

male

Details on test animals and environmental conditions:

Test animals
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 290-418 g
- Housing: Makrolon type-4 cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week

Environmental conditions
- Temperature (°C): 22 ±3°C
- Humidity (%): 30-70%
- Air changes (per h): 10-15
- Photoperiod (h dark / h light): 12h/12h

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

undiluted

Day(s)/duration:

Week 1, Week 2 and Week 3, 6 h exposure each week

Adequacy of induction:

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

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: bi-distilled water

Concentration / amount:

50%

Day(s)/duration:

2 weeks after last induction

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: bi-distilled water

Concentration / amount:

25 and 50% (Rechallenge test) purified test substance

Day(s)/duration:

2 weeks after challenge test

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group: 20 animals
Control group: 10 animals

Details on study design:

Irritation screening test:
An irritation screening test was performed to determine the minimal irritating concentration used in the induction period and the highest non-irritating concentration used for the challenge. Four different concentrations (5%, 25%, 50%, 100% diluted with bi-distilled water) were used on each animal for a 6-h exposure period. 4 guinea pigs were used. Application sites were assessed after 24 and 48 h. The most representative concentration to stimulate a state of immune hypersensitivity was 100 % used in the induction phase and a concentration of 50% was used in the challenge as the highest non-irritating concentration.

Main study
1) Induction
The fur was clipped from the left shoulder of each test animal and the patches applied, over a period of 3 weeks. The animals were treated with the test substance applied undiluted. Each animal received one patch per week which remained in place for approximately 6 h each. The repeated application was performed at the same site. The interval between exposure was one week. The control animals remained untreated. After the last induction exposure the test animals were left untreated for 2 weeks before the challenge. The skin responses were graded 24 h after the patches had been removed. Any gross skin reactions were recorded without depilation.

2) Challenge- performed on test Day 29
The animals previously exposed during the induction period (i.e. test group) as well as the previously untreated control animals were challenged two weeks after the last induction exposure using the test substance at 50 % in bi-distilled water. The fur was clipped from the left posterior quadrant of the side and back of the animals. The exposure period was 6 h on a naive skin site. The responses were graded at 24 and 48 h after the patches had been removed.

3) Second challenge
The test group was rechallenged 14 d following primary challenge. All animals in the test group were included in the rechallenge. The test substance was applied on the right cranial flank at 50 % in bi-distilled water and on the the right caudal flank at 25 % in bi-distilled water. The grading method used for irritation screen, induction and challenge was identical. The scoring system was performed by visual assessment of erythema, oedema and other clinical changes in skin conditions. They were assessed as follows:

0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Grading of all animals was done by positioning each animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830).

Challenge controls:

During induction the control animals remained antreated. At the challenge the controls were treated the same as the test group. Only the test group was challenged a second time.

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole

Results and discussion

Positive control results:

Five out of 10 test animals were observed with discrete/patchy erythema at the 24-h reading and moderate/confluent erythema was observed at the 48-h reading in all ten test animals after the challenge treatment with the highest tested non-irritating concentration of 2-Mercaptobenzothiazole at 0.03 % in mineral oil. No skin reactions were observed in the control group.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25% test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25% test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

bi-distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

bi-distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.03% 2-Mercaptobenzothiazole in mineral oil

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

discrete/patchy erythema

Remarks on result:

positive indication of skin sensitisation

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.03% 2-Mercaptobenzothiazole in mineral oil

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

moderate/confluent erythema

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

In the first challenge application 12 out of 20 animals reacted with discrete erythema after 24 and 48 h. the challenge was repeated with a purified sample in the sensitized animals. None of the animals reacted to the purified sample. The purification step is always applied to the commercial product.

Applicant's summary and conclusion

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-sensitising to the skin.

Executive summary:

A study was conducted to determine skin sensitisation potential of test substance, sodium cocoyl glutamate, using the Buehler method, according to OECD Guideline 406, in compliance with GLP. The test was performed in 20 (10 test and 10 control) male Himalayan spotted guinea pigs. Based on the screening test, 100% (undiluted) and 50% (in bi-distilled water) test substance concentrations were selected for induction and challenge phase, respectively. In the induction phase, fur was clipped from the left shoulder of each test animal and occlusive patches of the undiluted test substance applied (one a week at the same site) for 6 h over a period of 3 weeks. The skin responses were graded 24 h after the patches had been removed. Any gross skin reactions were recorded without depilation. The control animals remained untreated. After a rest period of 2 weeks, both the test and control group animals were challenged on a naive skin site with 50% test substance in bi-distilled water under similar conditions. The responses were graded at 24 and 48 h after the patches had been removed. All animals in the test group were additionally re-challenged on the right cranial flank at 50% in bi-distilled water and on the right caudal flank at 25% in bi-distilled water. The scoring system was performed by visual assessment of erythema, oedema and other clinical changes in skin conditions. They were assessed as: 0 = no visible change; 1 = discrete or patchy erythema; 2 = moderate and confluent erythema; 3 = intense erythema and swelling. Grading of all animals was done by positioning each animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830). During challenge 12 out of 20 animals reacted with distinct erythema. Therefore, a purified sample was prepared (which is equivalent to the commercial form) for the re-challenge procedure. During re-challenge with the purified sample no skin reactions were observed after 24 and 48 h in any animal. In the positive control group, five out of ten test animals were observed with discrete/patchy erythema at the 24-h reading and moderate/confluent erythema was observed at the 48-h reading in all ten test animals after the challenge treatment with the highest tested non-irritating concentration of 2-Mercaptobenzothiazole at 0.03 % in mineral oil. No skin reactions were observed in the negative control group. Under the study conditions, the test substance was determined to be non-sensitising to the skin (BASF, 2001).