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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Buehler test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 07, 2001 to August 08, 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The skin sensitisation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Conducted before the requirement of LLNA method was implemented.
Species:
guinea pig
Strain:
Himalayan
Remarks:
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: 290-418g
- 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
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12h/12h
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Induction: 100% (substance was applied undiluted)
Challenge: 50% diluted in water
Rechallenge: 25%, 50% diluted in water
Day(s)/duration:
6 hours once a week for 3 weeks
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled
Concentration / amount:
Induction: 100% (substance was applied undiluted)
Challenge: 50% diluted in water
Rechallenge: 25%, 50% diluted in water
Day(s)/duration:
6 hours
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 hours.
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 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.

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

3. 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 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
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.
Key result
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
Key result
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
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.03%
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.03%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

In the first induction, undiluted material was used and during the challenge, 12 out of 20 animals reacted with discrete erythema after 24 and 48 hours. 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.

Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the substance was not sensitizing to Guinea pig skin.
Executive summary:

A study was conducted to determine the in vivo skin sensitisation potential of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts according to OECD Guideline 406, in compliance with GLP. The experiment was performed on male Guinea pigs. For the induction phase, the undiluted test substance was used. During challenge, 12 out of 20 animals reacted with distinct erythema. Therefore, a purified/diluted sample (25%) was prepared for the re-challenge procedure. During the re-challenge with the purified sample, no skin reactions were observed after 24 and 48 h in any animal. Under the study conditions, the substance was not sensitizing to Guinea pig skin (RCC Ltd, 2001).

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Hypoallergenicity Test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From November 10, 2000 to November 1, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: Hypoallerginicity (T12C)
Version / remarks:
see 'Principles of method if other than guideline'
Deviations:
no
Principles of method if other than guideline:
Hypoallerginicity (T12C):
The method consists in an occlusive application of the test substance by means of Finn Chambers (alluminium cells of some 20 microlitres volume) on 20 selected subjects’ back or forearm, for 48 hours. After the removal of the occlusion, the cutaneous reactions induced by the product are evaluated. These evaluations are performed 24 and 48 hours later.
The repeated control of the treated areas allows us to trace the presence and/or the activity in elicitation of cutaneous reactions of common allergens.
GLP compliance:
yes
Remarks:
and GCP
Type of study:
other: Test T12C: Hypoallergenicity
Justification for non-LLNA method:
Conducted before the requirement of LLNA method was implemented.
Specific details on test material used for the study:
Appearance: liquid and transparent
Species:
other: Human
Sex:
male/female
Details on test animals and environmental conditions:
- 20 subjects
- Cleaned from sebum
No. of animals per dose:
Total of 20 persons
Details on study design:
- The method consists in an occlusive application of 48h. The test substance was put in Finn Chambers (aluminium cells with a diameter of 8 mm, 50 mm2 area, volume of 20 microlitres), which were filled slightly more than half their volume. The cells were applied on the back or on the forearm of the subject, on healthy skin previously cleaned from sebum.
- The test substance was tested at 7% in S.A.L.
Challenge controls:
Negative (empty cell) and vehicle controls
Positive control substance(s):
no
Key result
Reading:
other: after 24 and 48h
Group:
test chemical
Dose level:
7% in S.A.L.
No. with + reactions:
0
Total no. in group:
0
Clinical observations:
no effects
Remarks on result:
no indication of skin sensitisation
Key result

Results recording:

48 hours after application the strips were removed and then, after 30 minutes, the occlusive cutaneous reaction was evaluated (T1). An evident redness after 30 minutes was a possible sign of irritation caused by the product. A second check was made 24 hours after the removal (T2) of the plaster strip and a third one 48 hours later (T3). There are 5 kinds of reactions (1 -5); ranging from "no erythema to "possibly exudation and infiltration".

Interpretation of the results:

- The first check does not enable a final assessment; however, it provides useful indications:

1) A positive reaction (+, ++, +++) disappearing within the some hours after the removal of the cells is not regarded as an allergic reaction and therefore it is not included in the final calculation of the data. However, such reactions will be reported, since they can be regarded as either irritant reactions or a sign of moderate sensitivity of the subject to one or more components of the product.

2) A doubtful reaction (slightly visible +/-), which disappears within 24 hours after the removal of the cells is not included in the calculation.

- The checks made at successive times enabled identification of positive allergic reactions. The allergenic potential of the product is expressed as a percentage of allergic reactions and is evaluated considering: the number of reactions observed severity, type and duration of such reactions. On the basis of the data reported in literature, the presence/activity of common allergens in the product can be related to the percentage of the allergic reactions observed (from "no reaction" to "maximum").

Results:

The number of total allergic reactions and the percenrage of allergic reactions = 0.

Interpretation of results:
other: CLP criteria not met
Conclusions:
Under the study conditions, the substance was considered to be non-sensitizing to human skin.
Executive summary:

A supporting study was conducted to determine the skin sensitizing potential of the substance according to the Method T12C "Hypoallergenicity", in compliance with GLP. The method consisted of an occlusive application of the test substance (7% in S.A.L.) for 48 h by means of Finn Chambers (alluminium cells of 20 microlitres volume) on 20 selected subjects back or forearm. After the removal of the occlusion, the cutaneous reactions induced by the product were evaluated. These evaluations were performed 24 and 48 hours later. The checks made at successive times enabled identification of positive allergic reactions. The allergenic potential of the product was expressed as a percentage of allergic reactions and was evaluated considering the number of reactions observed, severity, type (from "no erythema to "possibly exudation and infiltration") and duration of such reactions. On the basis of the data reported in literature, the presence/activity of common allergens in the product can be related to the percentage of the allergic reactions observed (from "no reaction" to "maximum"). The number of total allergic reactions and the percenrage of allergic reactions were 0. Under the study conditions, the substance was considered to be non-sensitizing to human skin (Sirigu, 2000).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In vivo:

A study was conducted to determine the in vivo skin sensitisation potential of the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts according to OECD Guideline 406, in compliance with GLP. The experiment was performed on male Guinea pigs. For the induction phase, the undiluted test substance was used. During challenge, 12 out of 20 animals reacted with distinct erythema. Therefore, a purified/diluted sample (25%) was prepared for the re-challenge procedure. During the re-challenge with the purified sample, no skin reactions were observed after 24 and 48 h in any animal. Under the study conditions, the substance was not sensitizing to Guinea pig skin (RCC Ltd, 2001).

A supporting study was conducted to determine the skin sensitizing potential of the substance according to the Method T12C "Hypoallergenicity", in compliance with GLP. The method consisted of an occlusive application of the test substance (7% in S.A.L.) for 48 h by means of Finn Chambers (alluminium cells of 20 microlitres volume) on 20 selected subjects back or forearm. After the removal of the occlusion, the cutaneous reactions induced by the product were evaluated. These evaluations were performed 24 and 48 hours later. The checks made at successive times enabled identification of positive allergic reactions. The allergenic potential of the product was expressed as a percentage of allergic reactions and was evaluated considering the number of reactions observed, severity, type (from "no erythema to "possibly exudation and infiltration") and duration of such reactions. On the basis of the data reported in literature, the presence/activity of common allergens in the product can be related to the percentage of the allergic reactions observed (from "no reaction" to "maximum"). The number of total allergic reactions and the percenrage of allergic reactions were 0. Under the study conditions, the substance was considered to be non-sensitizing to human skin (Sirigu, 2000).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the in vivo skin sensitisation study with the read-across substance L-glutamic acid, N-coco acyl derivs., monosodium salts

and a supporting study with the substance, no classification for skin sensitisation is warranted according to EU CLP (1272/2008) criteria.