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Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
points 8.1 and 8.2 of Annex VIII of REACH have been amended. Nevertheless, adequate information from existing in vivo studies can still be used to fulfil the information requirement at any tonnage level.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From the 3th of April to the 4th May, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to internationally accepted guideline
Justification for type of information:
points 8.1 and 8.2 of Annex VIII of REACH have been amended. Nevertheless, adequate information from existing in vivo studies can still be used to fulfil the information requirement at any tonnage level.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
points 8.1 and 8.2 of Annex VIII of REACH have been amended. Nevertheless, adequate information from existing in vivo (non LLNA) studies can still be used to fulfil the information requirement at any tonnage level.
Species:
guinea pig
Strain:
Himalayan
Sex:
male/female
Details on test animals and environmental conditions:
Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Fullinsdorf
Number of animals: 15 males, 15 females (5 males and 5 females as control)
Age at start of treatment: male 7 weeks, females 8 weeks
Body weight at start of or treatment: male 266-317 g, females 264-363 g.
Identification: by unique cage number and corresponding ear tags.
Randomization: at the time of delivery
Acclimatization: one week under test conditions after veterinary examination.

HUSBANDRY
Room No: 135
Standard Laboratory Conditions.
Air-onditioned with 10-15 air changes per hour and hourly monitored environment with temperature 22±3 °C, relative humidity 40-70%, 12 hours
artificial fluorescent light/12 hours dark, music/light period.
Accomodation: Individually in Nakrolon type 3 cages with standard softwood bedding (‘Ligno cel’, Schill AG, CH—4132 Nuttenz).
Diet: Pelleted standard Kliba 342, Batch 57190 guinea pig breeding/ maintenance diet (‘Kliba’, Klingentalmuhle AG, CH—4303 Kaiseraugst), ad libitum. Analysis for contaminants were performed.
Water: community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid via the drinking water. Analysis for contaminants were porformed.

No necropsy was performed in the animals killed at termination o observation. All animals were killed at the end of the test period with an intraperitoneal injection of T61 (Hoechst AG) and discarded.
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
Physiological saline was used as vehicle for the intracutaneous and epicutaneous applications.
Concentration / amount:
PRELIMINARY STUDY
Intradermal injection
concentrations of 5 %, 3 % and 1 % of the test article in physiological saline. An additional intradermal pretest was performed on a further two guinea pigs at concentrations of 0.5 %, 0.3 % and 0.1 % of the test article in physiological saline.
Epidermal application
concentrations of 25 %, 15 %, 10 % and 5 % of the test article in physiological saline.

MAIN STUDY
The test article diluted to 0.5 % with physiological saline.
Intradermal injection
15 % in physiological saline
Epidermal application
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Remarks:
Physiological saline was used as vehicle for the intracutaneous and epicutaneous applications.
Concentration / amount:
PRELIMINARY STUDY
Intradermal injection
concentrations of 5 %, 3 % and 1 % of the test article in physiological saline. An additional intradermal pretest was performed on a further two guinea pigs at concentrations of 0.5 %, 0.3 % and 0.1 % of the test article in physiological saline.
Epidermal application
concentrations of 25 %, 15 %, 10 % and 5 % of the test article in physiological saline.

MAIN STUDY
The test article diluted to 0.5 % with physiological saline.
Intradermal injection
15 % in physiological saline
Epidermal application
No. of animals per dose:
10 males
10 females
Details on study design:
PRELIMINARY STUDY
Intradermal injections:
Intradermal injections (0.1 mL/site) were made into the clipped flank of two guinea-pigs at concentrations of 5 %, 3 % and 1 % of the test article in physio logical saline. An additional intradermal pretest was performed on a further two guinea pigs at concentrations of 0.5 %, 0.3 % and 0.1 % of the test articlein physiological saline, The resulting dermal reactions were assessed 24 hours later.
Epidermal appIIcations:
Patches of filter paper (2 x 2 cm) were saturated with concentrations of 25 %, 15 %, 10 % and 5 % of the test article in physiological saline and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and irm1y secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact or the test article. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema on a numerical basis according to the scale described above. Further examination or the sites were performed 24 and 48 hours after removal of the dressings.
Prior to the readings, the test sites were depilated, to clean the application sites From staining produced by the test article.
The allocation of the different test sites on the animals was alternated in order to minimize site to site variation in responsiveness.

MAIN STUDY
Intradermal Injections:
An area or dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free or hair, Three pairs or intradermal injections (0.1 mL/site) were made at the border o a 4 x 6 cm area in the clipped region as follows:
Test group:
1) Freund’s complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 0.5% with physiological saline.
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freund’s complete adjuvant and the vehicle used in (2).
Control Group:
1) Freund’s complete adjuvant 50:50 with bi-distilled water.
2) Vehicle used in (2) for test group.
3) Freund’s complete adjuvant 50:50 with bidistilled water.
Epi dermal appi i cati ons:
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 physiological saline) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours The epidermal application procedure described ensured intensive contact of the test article.
The guinea-pigs of the control group were treated as described above with the omission of test article.
Reaction sites were assessed for erythema and edema immediately, 24 and 48 hours after removal or the dressing, using the numerical grading system described previously.
Challenge controls:
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 physiological saline) of the test article and
b) with the vehicle only and applied to the (a) left flank and (b) right flank using the same method as for the epidermal application.
The dressings were removed approximately 24 hours later. The sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of thedressing, using the numerical scoring system as described under preliminary study.
Prior to the first reading the test sites were depilated to facilitate the evaluation of a possible erythema reaction.
Positive control substance(s):
yes
Remarks:
A control group (Formaldehyde solution) is tested twice a year for sensitivity check of the guinea pig strain.
Positive control results:
MAIN TEST
No positive reactions were evident after the first challenge application, neither when treated with the vehicle alone nor when treated with the test article.

SYMPHOMS
Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 6; necroses were observed from day 7 to 11 and desiccation from day 12 to 21 and exrollatlon from day 22 to 23 (termInation of the study).
Staining was observed on the skin area after rirst challenge application from day 23 to 25.
Reading:
1st reading
Group:
negative control
Total no. in group:
5
Clinical observations:
physiological saline solution
Remarks on result:
other: Reading: 1st reading. Group: negative control. Total no. in groups: 5.0. Clinical observations: physiological saline solution.
Reading:
1st reading
Group:
positive control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Erythema and oedema on the application site, necroses and desiccation, exfoliation
Remarks on result:
other: Reading: 1st reading. Group: positive control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Erythema and oedema on the application site, necroses and desiccation, exfoliation.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
erythema, oedema, necroses, dessication, exfoliation, staining
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: erythema, oedema, necroses, dessication, exfoliation, staining.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
erythema, oedema, necroses, dessication, exfoliation, staining
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: erythema, oedema, necroses, dessication, exfoliation, staining.

PRE-TEST

Intradermal injection: according to Magnusson Kligman and to the rindings observed, the concentration selected for the main study was 0.5%.

Epicutaneous application: according to Magnusson Kligman, and to the findings observed, the concen tration selected for the induction period was 15% and for the challenge procedure 10%.

MAIN TEST

Seven out of 20 and five out of twenty animals showed erythema at the 24 and 48-hour reading, respectively, when treated with a 10% test article dilution in physiological saline.

No positive reactions were observed on the flanks treated with physiological saline alone.

No death occurred during the study.

SYMPHTOMS

Application area around the injection site 1 was found to show erythema and edema from day 2 to 6; necroses were observed from day 7 to 21 and desiccation from day 22 to 25. Around the injection sites 2 and 3, erythema and edema were observed from day 2 to 6 and staining from day 2 to 11.

Application area around the injection site 2 showed necroses on days 7 to 10, desiccation from day 8 to 16 and exfoliation from day 12 to 21.

Fissures were observed from day 22 to 25.

Around the injection site 3, necroses were observed from days 7 to 21 and desiccation from day 22 to 25. In addition epidermal induction and first challenge application area showed staining from day 10 to 12, respectively 23 to 25. On day 9 of test no observation could be performed because the animals were treated semi-occlusively.

No systemic symptoms were observed during the study.

The body weight gain of the animals was not afrected adversely during the study.

Interpretation of results:
sensitising
Remarks:
Classification criteria according to the CLP Regulation 1272/2008 and its amendments
Conclusions:
From the results described above moderate allergenic potency of the test article was concluded. The results were interpreted according to the rating or Magnusson and Kligman (1970). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983, this test article is considered to be a sensitizer.
Executive summary:

The allergenic potential of the substance on albino guinea pigs was evaluated in a Maximization Test of B. Magnusson and A.M. Kligman (1970), according to the OECD Guideline 406 and the EU Method B.6 (Skin sensitization).

Ten animals (5 males, 5 females) were used as control group and 20 animals (10 males, 10 females) were used as test group.

Prior to the first reading of the reactions, the skin was depilated to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time. Due to the unequivocal findings observed after the first challenge, no second challenge was performed.

The highest non irritating concentration used for challenge application was 10 %.

No toxic symptoms were evident in the guinea pigs or neither the control nor in the test group. No death occurred.

From the results described above moderate allergenic potency of the test article was concluded. The results were interpreted according to the rating or Magnusson and Kligman (1970). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983, this test article is considered to be a sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The allergenic potential of the substance on albino guinea pigs was evaluated in a Maximization Test of B. Magnusson and A.M. Kligman (1970), according to the OECD Guideline 406 and the EU Method B.6 (Skin sensitization).

Ten animals (5 males, 5 females) were used as control group and 20 animals (10 males, 10 females) were used as test group.

Prior to the first reading of the reactions, the skin was depilated to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time. Due to the unequivocal findings observed after the first challenge, no second challenge was performed.

The highest non irritating concentration used for challenge application was 10 %.

No toxic symptoms were evident in the guinea pigs or neither the control nor in the test group. No death occurred.

From the results described above moderate allergenic potency of the test article was concluded. The results were interpreted according to the rating or Magnusson and Kligman (1970). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983, this test article is considered to be a sensitizer.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

According to the CLP Regulation n. 1272/2008, a substances shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub- categorisation (1A and 1B) in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test (according to 3.4.2.2.4.1).

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, 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.

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

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test- ≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

Based on animal test (non-adjuvant Guinea pig test method) results performed classified in subcategory 1A/1B because the positive reaction is < 30 %.

The substance could be classified according to CLP Regulation n. 1272/2008 as a Skin sensitizer, category 1, Hazard statement H317, due to the percentage of positive response (15%) observed in animal test.

However, the study reported was performed according to OECD 406 Magnusson test, that includes the use of the adjuvant.

This test has been used for over 40 years, to detect the sensitising potential of chemicals through a test system maximizing the sensitivity by both intradermal and epidermal induction and use of an adjuvant (Freund’s Complete Adjuvant).

When an adjuvant type guinea pig test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

Seven out of 20 animals (35%) and five out of 20 animals (25%) showed erythema at the 24 and 48 hours readings.

Based on these results, and in a conservative way (25 % vs 30%), according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the substance shall be classified as a skin sensitizer, H317, category 1B.