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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:
7 May 2001 to 23 August 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

Constituent 1
Chemical structure
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:
C17 H32 N1 O4 Na1
IUPAC Name:
l-Glutamic acid, N-coco acyl derivs., monosodium salts

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 290 - 418 g
- Housing: Makrolon type-4 cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Induction: 100% (substance was applied undiluted)
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Challenge: 50% diluted in water
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Rechallenge: 25%, 50% diluted in water
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-hour 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
- 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 material applied undiluted. Each animal received one patch per week which remained in place for approximately 6 hours 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 hours after the patches had been removed. Any gross skin reactions were recorded without depilation.

- 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 material 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 hours on a naive skin site. The responses were graded at 24 and 48 hours after the patches had been removed.

- Second challenge
The test group was rechallenged 14 days following primary challenge. All animals in the test group were included in the rechallenge. The test material 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-hour reading and moderate/confluent erythema was observed at the 48-hour 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)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none

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 hours. The challenge was repeated with a purified sample in the sensitised 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 sensitising according to EU criteria
Conclusions:
During challenge and re-challenge, distinct signs for sensitisation were observed with the test material. Therefore, a second rechallenge was performed using a purified test material sample. With this sample no effects were observed. The commercial product is equivalent to the purified test sample.
Executive summary:

The test material has been investigated for its sensitisation potential in accordance with the standardised guideline OECD 406 (Buehler protocol), under GLP conditions.

For the induction phase undiluted material was used. During challenge 12 out of 20 animals reacted with distinct erythema. Therefore, a purified sample has been prepared for the re-challenge procedure. During re-challenge with the purified sample no skin reactions were observed after 24 and 48 hours in any animal.The registered substance is not considered to be sensitising because the purification step is applied to all commercial material.