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Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across based on grouping of substances (category approch)
Adequacy of study:
key study
Study period:
From July 25 to September 8, 1989.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed according to GLP but used in RA Justification for Read Across is reported iin the Category Justification Report attached to the Section 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted May 12, 1981
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was performed before the validation of LLNA OECD method and is considered to be valid and acceptable for the assessment.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 7 to 8 weeks, males and females respectively.
- Weight at study initiation: males 359-454 g, females 390-450 g.
- Housing: individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).
- Diet: ad libitum, pelleted standard Kliba 342.
- Water: ad libitum, community tap water.
- Acclimation period: one week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 to 70 %
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark, music/light period.
Route:
intradermal and epicutaneous
Vehicle:
other: bi-distilled water for intracutaneous applications and petrolatum oil for epicutaneous applications.
Concentration / amount:
Intradermal injection of test article diluted to 1 %, dermal application of 1 5 % (in petrolatum oil) and dermal challenge with 10 % (in petrolatum oil).
Route:
epicutaneous, occlusive
Vehicle:
other: bi-distilled water for intracutaneous applications and petrolatum oil for epicutaneous applications.
Concentration / amount:
Intradermal injection of test article diluted to 1 %, dermal application of 1 5 % (in petrolatum oil) and dermal challenge with 10 % (in petrolatum oil).
No. of animals per dose:
15 males and 15 females: 10 x sex in treatment group and 5 per sex in control group.
Details on study design:
TEST ARTICLE PREPARATION
The test article and vehicle were placed into a glass beaker on a tared Mettler PK 300 balance. A weight/weight dilution was prepared using a homogenizer. Homogeneity of the test article in vehicle (bi-distilled water for intracutaneous applications and petrolatum oil for epicutaneous applications) was maintained during treatment using a magnetic stirrer. The preparations were made immediately (for second epidermal pretest not stated in raw data) prior to each dosing.

PRELIMINARY STUDY
The objective of this investigation was to identify irritant test article concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.

Intradermal injections: intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5 %, 3 %, 1 %, 0.5 %, 0.3 % and 0.1 % of the test article in bi-distilled water. The resulting dermal reactions were assessed 24 hours later.

Epidermal applications: patches of filter paper ( 2 x 2 cm) were saturated with concentrations of 25 %, 15 %, 10 % and 5 % of the test article in petrolatum oil and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wound round the trunk and covered with impervious adhesive tape. This procedure ensured intensive contact of the test article with the guinea pig skin. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and oedema on a numerical basis according to the scale described above. Further examination of the sites was performed 24 and 48 hours after removal of the dressings.
An additional epidermal pretest was performed one week after the first epidermal pretest with test article concentrations of 15 %, 10 %, 5 % and 1 % (petrolatum oil).

MAIN STUDY
A. INDUCTION EXPOSURE
A.1 - Intradermal injections: an area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
1) freunds' complete adjuvant 50:50 with bi-distilled water for injection.
2) the test article, diluted to 1 % with bi-distilled water.
3) the test article at the concentration of 1 %, emulsified in a 50:50 mixture of Freunds' complete adjuvant and the bi-distilled water.
- Control: treated accordingly with the omission of the test article.

A.2 - Epidermal applications: one week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (15 % in petrolatum oil) and placed over the injection sites of the test animals. The patch was covered by aluminium foil and firmly secured by an elastic plaster wound round the trunk of the animal and secured with impervious adhesive tape.
The dressings were left in place for approximately 48 hours.
- Control: the guinea-pigs of the control group were treated as described with petrolatum oil only.
- Assessment: the reaction sites were assessed for erythema and oedema immediately, 24 and 48 hours after removal of the dressing.

B. CHALLENGE EXPOSURE
B.1 - Challenge: the test and control guinea-pigs were challenged two weeks after the epidermal induction application.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig. Two patches ( 2 x 2 cm) of filter paper were saturated with:
a) non-irritant concentration (10 % in petrolatum oil) of the test article and
b) with the vehicle only
applied to the left (a) flank and right (b) flank respectively using the same method as for the epidermal application.
The dressings were removed approximately 24 hours later.
- Assessment: the sites were assessed for erythema and oedema immediately, 24 and 48 hours after removal, using the numerical scoring system as described previously.
- Control: the control animals were treated in the same way as described above.

B.2 - Re-challenge: a second challenge was performed two weeks after the first challenge.
The method for the animals of the test article-treated group was equal to that described for the first challenge with the exception that the flanks of all the guinea-pigs were changed (a - vehicle; b - test article dilution).
- Control: the control animals were treated with the vehicle only on the left flank.

READINGS AND SCORING
The following parameters were recorded:
Erythema (E) - 0 to 4 numerical scores
Oedema (0) - 0 to 4 numerical scores
Diameter (D) - mm

Erythema and oedema were assessed using the following numerical grading system:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema (barely perceptible) 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimeter) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4
Positive control substance(s):
yes
Remarks:
formaldehyde solution
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
Erythema reaction.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: Erythema reaction..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
Erythema reaction.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: Erythema reaction..
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
6
Total no. in group:
4
Clinical observations:
none
Remarks on result:
other: A control group (Formaldehyde solution) is tested twice a year for sensitivity check of guinea pig strain. This test was run in April 1989

PRETEST

During pretest, the following reactions were observed:

INTRADERMAL INJECTION

Vehicle: bi-distilled water

Animal N. Sex Conc. % Reaction readings after 24 hrs
Erythema Oedema Diameter
196 M 5 2 2 12 x 12
197 F 5 2 2 12 x 15
196 M 3 2 2 11 x x13
197 F 3 2 2 11 x 11
196 M 1 1 1 7 x 8
197 F 1 1 1 5 x 5
196 M 0.5 1 0 4 x 4
197 F 0.5 1 0 6 x 6
196 M 0.3 1 0 4 x 4
197 F 0.3 1 0 4 x 4
196 M 0.1 1 0 4 x 4
197 F 0.1 1 0 4 x 4

The concentration selected for the main study was 1 %.

EPIDERMAL APPLICATION

Vehicle: petrolatum oil

Animal N. Sex Conc. % Reaction readings after removal of bandage
Immediately After 24 hrs After 48 hrs
Erythema Oedema Erythema Oedema Erythema Oedema
198 M 25 0 0 0 0 0 0
15 0 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
199 F 5 0 0 0 0 0 0
25 1 0 0 0 0 0
15 0 0 0 0 0 0
10 0 0 0 0 0 0
200 M 10 0 0 1 0 0 0
5 0 0 1 0 0 0
25 1 0 1 0 0 0
15 0 0 1 0 0 0
354 F 15 0 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
25 0 0 0 0 0 0

The concentration selected for the induction period was 15 %.

EPIDERMAL APPLICATION

Vehicle: petrolatum oil

Animal N. Sex Conc. % Reaction readings after removal of bandage
Immediately After 24 hrs After 48 hrs
Erythema Oedema Erythema Oedema Erythema Oedema
326 F 15 1 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
1 0 0 0 0 0 0
327 F 1 0 0 0 0 0 0
15 0 0 0 0 0 0
10 0 0 0 0 0 0
5 0 0 0 0 0 0
328 F 5 0 0 0 0 0 0
1 0 0 0 0 0 0
15 0 0 0 0 0 0
10 0 0 0 0 0 0
329 F 10 0 0 0 0 0 0
5 0 0 0 0 0 0
1 0 0 0 0 0 0
15 0 0 0 0 0 0

The concentration selected for the challenge procedure was 10 %.

MAIN TEST

Control group

No positive reactions were evident after the first and second challenge application neither on the right nor on the left flank.

Test group

First Challenge - Erythema was observed in five out of twenty animals (25 %) at the 24-hours reading and three out of twenty animals (15 %) at the 48-hours reading when treated with a test article concentration of 10 %.

Second Challenge - No positive findings were observed after the second challenge application in any animal.

VIABILITY / MORTALITY

No death occurred during the entire test period.

SYMPTOMS, LOCAL

Control group

Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 8; Necroses from day 10 to 32 and exfoliation from day 33 to 39 (termination of test). Application area around the injection site 2 was found to show erythema and edema from day 2 to 4.

Test group

Application area around the injection sites 1 and 3 was found to show erythema and oedema from day 2 to 8; necroses from day 10 to 32 and exfoliation from day 33 to 39 (termination of test). Application area around the injection site 2 was found to show erythema and oedema from day 2 to 8.

Epidermal application area for induction showed discoloration from day 10 to 14.

Discoloration was also observed on first challenge application area from day 23 to 32 and on second challenge application area from day 37 to 39 (termination of test).

On day 9 of the test no observation could be performed because the animals were treated semi-occlusively.

SYMPTOMS, SYSTEMIC

No systemic symptoms were observed during the entire test period.

BODY WEIGHTS

Males no. 267 and 270 and female no. 280 lost weight during acclimatization period and females no. 277 and 289 lost weight during treatment period.

The body weight gain of the remaining animals was not affected during the entire test period.

Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
Not sensitising.
Executive summary:

Method

The purpose of this skin sensitization study was to assess the allergenic potential of test item when administered to the skin of male and female albino guinea pigs. For this purpose the Maximization, according to the OECD guideline 406, was used. Ten animals (5 males, 5 females) were used as control group and twenty animals (10 males, 10 females) were used as test group.

Prior to the first reading of the reactions, the skin was washed-off with the vehicle petrolatum oil to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time.

Results

The highest non-irritating concentration used for first and second challenge was 10 %.

First Challenge - Erythema was observed in five out of twenty animals (25 %) at the 24-hours reading and three out of twenty animals (15 %) at the 48-hours reading when treated with a test article concentration of 10 %.

Second Challenge - No positive findings were observed after the second challenge application in any animal.

Conclusion

According to CLP Regulation (EC1272/2008) a substance in considered a skin sensitizer when the Guinea pig maximisation test results indicate that a number of animals ≥ 30 % to < 60 % responding at concentrations > 0.1 % to ≤ 1 % of intradermal induction dose or a number of animals ≥ 30 % responding at > 1 % intradermal induction dose. Less than the 30 % of animals showed a reaction, therefore the substance can be considered as non sensitising.

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

A sensitization test in albino guinea pigs was performed on the substance under registration (CAS 13863-31-5) to determine the contact allergenic potency of the test compound in albino guinea pigs. The optimization test was used, an intracutaneous sensitization procedure similar to the method recommended in the "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" (1959), the US Association of Food and Drug Officials (AFDO).

Under the experimental conditions employed, significant differences (P ≤ 0.05) between the test group and the vehicle treated controls were found. A skin sensitizing potential for test substance cannot be excluded, but the purity of the substance is unknown, therefore no reliable conclusion can be drawn from this experiment (Ciba-Geigy Ltd., 1975).

A further test was reported; it was performed according to OECD guideline 406 (Maximisation test on Guinea Pig) on the analogous dihydroxyethylamino derivative 3a-A(Na) belonging to the Stilbene Fluorescent Whitening Agents category. The liver metabolisation modelling, performed using the OECD Toolbox, indicates that the two substances share the common first pass metabolite monohydroxyethylamino.

Ten animals (5 males, 5 females) were used as control group and twenty animals (10 males, 10 females) were used as test group. Prior to the first reading of the reactions, the skin was washed-off with the vehicle petrolatum oil to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time. The highest non-irritating concentration used for first and second challenge was 10 %. First Challenge - Erythema was observed in five out of twenty animals (25 %) at the 24-hours reading and three out of twenty animals (15 %) at the 48-hours reading when treated with a test article concentration of 10 %. Second Challenge - No positive findings were observed after the second challenge application in any animal. Therefore, according to the CLP Regulation (EC 1272/2008), the substance can be considered as non sensitising (RCC, Research & Consulting Company AG., 1989).

Within the whole category, nine over fourteen registered substances covering at least one member per group (see data matrix in the Category Justification Report attached to the section 13 of the dossier) were tested and none of the existing tests arisen any concern for skin sensitisation.

All substances of the category were modelled using the OECD Toolbox and the provisional results about sensitisation and protein binding were calculated for all members. No alerts were reported for any substance. The same was performed for 3b-A, and a common behaviour regarding this end point within the category was observed. Based on all those considerations, the available study on the analogous substance can be considered as representative for the substance under registration, which can then be considered not sensitizing, too.

Skin adsorption was evaluated and calculated for all members of the category (see Category Justification Report, Section 13 of the dossier).

As it can be noted, the influence of the variability in functional group is very low, more related to the variability in the polarity of the substance than on potential reactivity that can arise a concern. From a metabolic point of view, an estimation with OECD Toolbox of the dermal metabolism was performed in order to verify if breakdown products could be formed. Skin adsorption is considered the condition for sensitisation to express, therefore no concern for sensitisation properties can be expected for all members of the category.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin sensitizer, on the basis of results from the Guinea pig Maximization test, are reported into the second adaptation to technical progress*. A substance in considered a skin sensitizer when:

- an adjuvant type test method for skin sensitisation is used and a response of at least 30 % of the animals is considered as positive;

- for a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive;

- a stimulation index of three or more is considered a positive response in the local lymph node assay.

Therefore, in the case of the Guinea pig maximisation test a substance is classify as sensitising if the results indicate that a number of animals ≥ 30 % to < 60 % responding at concentrations > 0.1 % to ≤ 1 % of intradermal induction dose or a number of animals ≥ 30 % responding at > 1 % intradermal induction dose.

Less than the 30 % of animals showed a reaction, therefore the substance can be considered as non sensitising.

In conclusion, the available experimental data are adequate for classification and labelling and the test substance is not classified as skin sensitizing according to CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures