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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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. The test was performed on a similar substance within the category of Stilbene Fluorescent Whitening Agents, the analogous dihydroxyethyl derivative disulphonated sodium salt. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted May 12, 1981
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

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.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
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).
Challengeopen allclose all
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

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
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 group
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 group
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 group
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.