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Administrative data

Description of key information

Based on the results of an open epicutaneus test in guinea pigs (BASF SE, 2002), supporting animal studies, and the evaluation of human data on skin sensitisation the test substance is considered to be sensitising to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: G. Klecak: Identification of Contact Allergens (Predictive Tests in Animals) in: Advances in Modern Toxicology Vol. 4, - Dermatotoxicology and Pharmacology - ed. F. N. Marzulli, H. I. Maibach - Pages 321 - 324
Version / remarks:
1977
Deviations:
not specified
GLP compliance:
yes
Type of study:
open epicutaneous test
Justification for non-LLNA method:
Study data with guinea pigs was available that was published before the LLNA had been adopted as standard requirement under REACH.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: PK 95/222

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
Species:
guinea pig
Strain:
other: (HA) BR [SPF]
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH - Wiga, Kisslegg, FRG
- Age at study initiation: Young adult animals
- Weight at study initiation: 333 - 395 g
- Housing: 4 animals were housed in Makrolon cages, type IV
- Diet (ad libitum): Kliba-Labordiaet 341 (Klingentakmuehle AG, Kaiseraugst, Switzerland).
- Water (ad libitum): tap water; about 2 g of ascorbic acid per 10 l water was added to the drinking water twice a week.
- Acclimation period: 6 days before the beginning of the study in the laboratory for dermal toxicity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
15 %, 4 %, 0.4 % and 0.15 % of the test substance
Day(s)/duration:
28 days
Adequacy of induction:
other: 0.4 % is the highest non irritant concentration.
Route:
epicutaneous, open
Vehicle:
water
No.:
#1
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
15 %, 4 %, 0.4 % and 0.15 % of the test substance in aqua bidest.
Day(s)/duration:
3
Adequacy of challenge:
other: 0.4 % is the highest non irritant concentration.
No.:
#2
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
15 %, 4 %, 0.4 % and 0.15 % of the test substance
Day(s)/duration:
3
Adequacy of challenge:
other: 0.4 % is the highest non irritant concentration.
No. of animals per dose:
8
Details on study design:
Pretest:
In a pretest, the irritancy profile was determined by applying 25 µL of various concentrations (0.15 %, 0.4 %, 4 %, 15 %, and 40 %) of aqueous test substance solutions to a 2-cm² area of the shaved flanks of 6 Guinea pigs. All test concentrations were each tested on the same test animal. Test sites were visually evaluated 24 h, 48 h and 72 h after application of test solutions. The dose not causing a reaction in any animal (maximum non-irritant concentration) and the dose causing a reaction in 25 % of the animals (minimum irritant concentration) were determined.

Induction:
20 inductions were conducted. During the induction phase of the main test, groups of 8 Guinea pigs were administered 100 µL of test solution to an 8-cm² area of the right flank skin, once daily on working days (5/wk) for four weeks : Gr 4: 15 % in aqua dest.; Gr 5: 4 %; Gr 6: 0.4 %; Gr 7: 0.15 %.
The control group animals (Gr. 1, 2 and 3) were not treated since the distilled water used as formulating agent was not expected to influence the results of the study. Skin readings were performed 24 h after application.

Challenge:
Three days after the last induction treatment, each test group animal and animals from control group 1 were challenged with 25 µL of 4 different concentrations (15 %, 4 %, 1.5 %, 0.4 %) to a 2-cm² area on the previously untreated flank. Skin reactions were read on an all-or-none basis at 24, 48 and 72 h after application of the solutions.
The second challenge treatment was performed 17 days after the last induction treatment on all treatment groups and on control groups 1 and 2, while control group 3 remained untreated. Skin reactions of test animals were compared with control animals after challenge.

Evaluation:
The test was considered to be positive if at least one guinea pig of the particular concentration group exhibited positive skin reactions with a non-irritant concentration 24 and/or 48 h and/or 72 h after application of the test substance, the control animals showing a negative reaction.

Reference: Klecak G.: "Identification of contact allergens (Predictive tests in animals)", in: Advances in Modern toxicology, Vol. 4 - Dermatotoxicology and Pharmacology (eds.: Marzulli FN & Maibach HI), p. 321-324, (1977).




Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
15 %
No. with + reactions:
7
Total no. in group:
8
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
4%
No. with + reactions:
4
Total no. in group:
6
Clinical observations:
2 animals died 35 days after the beginning of the study.
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.4 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
3 animals died 32, 34 and 35 days after the beginning of the study.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
0.15 %
No. with + reactions:
0
Total no. in group:
7
Clinical observations:
1 animal died 32 days after the beginning of the study.

Pretest:

In the pretest, a 15% test substance formulation (in aqua bidest) was found to be the minimum irritant concentration and the 4 % formulation the maximum non-irritant concentration. However, in the main test, 2 of 8 guinea pigs given 4 % test substance developed slight-moderate erythema and slight edema and scaling during the 3rd week of induction treatment. Thus, the maximum non-irritant concentration during induction was 0.4%.

Induction:

Individual results obtained during the induction phase:

- 3 out of 8 animals treated with 15 % test substance solution exhibited very slight to well-defined erythema during the first week of induction. After the 6th induction until the 19th induction all animals showed very slight to well-defined skin reactions, partially in addition to scaling and superficial scabbing.
- The 4 % concentration caused very slight to well-defined skin reactions and scaling in 2 out of 8 animals after the 12th induction until the 19th induction.
- No signs of skin reactions during the induction phase were seen at the application sites of the 0.4 and 0.15 % test substance concentrations.

Challenge:

- After the first challenge with the 15 % test substance concentration very slight to moderate skin reactions were caused, partially in addition to scaling and superficial scabbing in all animals induced with 15 %, 4 % and 0.4 % and in 4 out of 8 animals induced with 0.15 %. At the second challenge, all animals induced with 4 %, 0.4 % and 0.15 % showed positive reactions, as well as 7 out of 8 animals induced with 15 %.
- Challenge with 4% t
est substance concentration caused very slight to well-defined skin reactions in 4 out of 8 animals induced with 15 %, 4 % and 0.4 % and in 2 out of 8 animals induced with 0.15 %. At the second challenge, 6/8, 4/6, 4/5, 2/7 animals had positive reactions induced with 15 %, 4 %, 0.4 % and 0.15 %, respectively.
- Challenge with 0.4 %
test substance concentration did not cause any skin changes in all groups after the first challenge. However, after the second challenge 1 out of 8 animals induced with 0.4 % had a well-defined erythema.
- Challenge with 0.15 %
test substance concentration did not cause any skin changes neither after the first nor after the second challenge.

Under the experimental conditions of this study, the test substance was not sensitising in the Open Epicutaneous Test at challenge with concentrations of up to 0.4 % (max. non-irritant concentration) when induction was performed with concentrations lower than or equal to 4 %. The test substance was a skin sensitiser at 15 %.

The number of animals with skin findings after the 1st challenge and after the 2nd challenge.

 

Concentration of

(Test substance in aqua bidest.)

Induction

1st challenge

2nd challenge

15 %

4 %

0.4 %

0.15 %

15 %

4 %

0.4 %

0.15 %

Control group 1*

-

1/8

0/8

0/8

0/8

2/6

1/6

0/6

0/6

Control group 2

-

-

-

-

-

0/8

0/8

0/8

0/8

Test group 4

15%

8/8

4/8

0/8

0/8

7/8

6/8

1/8

0/8

Test group 5**

4%

8/8

4/8

0/8

0/8

6/6

4/6

0/6

0/6

Test group 6***

0.4%

8/8

4/8

0/8

0/8

5/5

4/5

0/5

0/5

Test group 7****

0.15%

4/8

2/8

0/8

0/8

7/7

2/7

0/7

0/7

 * 2 animals of control group 1 died 35 days after the beginning of the study.

 ** 2 animals of test group 5 died 34 days after the beginning of the study.

 *** 3 animals of test group 6 died 32, 34 and 35 days after the beginning of the study.

**** 1 animal of test group 7 died 32 days after the beginning of the study.

Macroscopic examination revealed that the cause of deaths was not substance-related.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

An open epicutaneous test in guinea pigs was identified as the key study (BASF SE, 1997). The test was performed in compliance with GLP based on the method of G. Klecak: Identification of Contact Allergens (Predictive Tests in Animals) in: Advances in Modern Toxicology Vol. 4, - Dermatotoxicology and Pharmacology - ed. F. N. Marzulli, H. I. Maibach - Pages 321 - 324 (1977).The open epicutaneous test investigated a range of concentrations during induction and challenge. The study concluded that HHT was not a sensitiser under specific test conditions. However the findings indicate that sensitisation potentially occurs when the specific conditions are exceeded.

It was concluded that the test substance was not sensitising at challenge with test substance concentrations of up to 0.4 % (max. non-irritant concentration) when induction was performed with concentrations lower than or equal to 4 %. However, the findings indicate that sensitisation potentially occurs when the specific conditions are exceeded (i.e.test substance was a skin sensitiser at 15 %). The skin sensitising potential of the test substance is supported by several reports on positive skin sensitisation reactions in humans after skin contact with the substance or products containing the substance (Schnuch et al., 1998; Roed-Petersen, 1977; de Groot et al., 1986; Dahl, 1981; Latorre et al. 2011). Taken together, the test substance is considered to be sensitising to the skin.

Supportingly, the substance was found to be a skin sensitiser in a murine local lymph node assay (L.A.C.S.A., 2015). In this study, an EC3 value could not be derived, since the S.I. values of the low and mid dose groups were above the threshold value of 3, and no conventional dose-response was observed. Further evidence for skin sensitising properties, including in humans, is available in public literature.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The Commission Working Group on the Classification and Labelling of Dangerous Substance, Meeting at ECB Ispra, 1997 (ECBI/32/97) agreed to maintain the skin sensitising classification and a 0.1 % concentration limit after evaluating both human and animal data relating to this test substance.

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance needs to be classified as skin sensitisng cat. 1 (H317) under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.