Registration Dossier

Administrative data

Description of key information

Two maximisation tests in guinea pigs were performed according to OECD 406 (or comparable) and GLP in 1989 and 1985. According to these both tests the test item is a skin sensitiser. An Klecak Open Epicutaneous Test in guinea pigs did not show any skin reactions.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 July - 25 Aug 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No reliability check and no irritation scores for the induction phase were reported.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
no reliability check and no irritation scores for the induction phase were reported
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in-vivo testing was set into force.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 275-350 g
- Housing: 2-3 animals per cage in opaque PPL cages (Type IV), pinewood sawdust was used as bedding material
- Diet: Altromin 3113 (Chr. Petersen Ltd, Jorløse, Jerslev, Denmark), ad libitum
- Water: Vitamin C enriched tap water, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal induction: paraffin oil; dermal induction: 70% ethanol
Concentration / amount:
intradermal: 1%, epicutaneous: 25%
Day(s)/duration:
intradermal induction: on Day 0; dermal induction: on Day 7
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: 70% ethanol
Concentration / amount:
2%
Day(s)/duration:
3 weeks after intradermal induction; exposure period: 24 h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, semiocclusive
Vehicle:
other: 70% ethanol
Concentration / amount:
0.2%
Day(s)/duration:
1 week after challenge
No. of animals per dose:
20 (controls), 20 (test group)
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed to determine the appropriate dose level of the test substance following intradermal and topical administrations. The intradermal irritancy of the test substance was investigated in order to find a slight to moderate irritant test substance concentration for the intradermal induction using intradermal injections as in the main study. For the intradermal administration test item concentrations of 0.63, 1.25, 2.5 or 5% were injected intradermal ten times in two animals each. Slight erythema was observed at 0.63%, moderate erythema at 1.25% and moderate to severe erythema with necrosis at 2.5 and 5% test substance.
The topical irritancy of the test substance was investigated in order to find a slight to moderate irritant test substance concentration for the dermal induction using closed patch tests as in the main study. For the topical administration test item concentrations of 50 and 100% were applied two times each to the skin of two animals. Moderate to severe erythema was observed at 50 and 100% test substance.
The topical irritancy of the test substance was investigated in order to find the highest non-irritant test substance concentration for the challenge application. For the topical administration test item concentrations of 0.25, 0.5, 1.0 and 2.0% were applied each with 3 patches to the skin of 3 animals. Additionally, test substance concentrations of 3.13, 6.25, 12.5 and 25% were applied each with 3 patches to the skin of further 3 animals. No skin reactions were observed up to and including 1.0% test substance concentration. At 2.0% test substance concentration, no skin reactions were observed in 2/3 animals and very slight erythema was observed in 1 animal. Slight to moderate erythema was noted at 3.13% test substance concentration. At 6.25 and 12.5% test substance concentration, moderate erythema was noted. Moderate to severe erythema was observed at 25% test substance concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture FCA/water
Injection 2: test substance in paraffin oil
Injection 3: equal amounts of 2% test substance and FCA
Epicutaneous: 25% test substance
- Control group:
Injection 1: a 1:1 mixture FCA/water
Injection 2: paraffin oil
Injection 3: equal amounts of paraffin oil and FCA
Epicutaneous: 70% ethanol
- Site: scapular region (intradermal + epicutaneous)
- Frequency of applications: every 7 days
- Duration: Days 0-7
- Concentrations: intradermal 1%, epicutaneous 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 2 (challenge and rechallenge)
- Day of challenge: 21 (challenge) and 28 (rechallenge)
- Exposure period: 24 h
- Test groups: 2% test substance and 70% ethanol (challenge); 0.2% test substance (rechallenge)
- Control group: 2% test substance and 70% ethanol (challenge); 0.2% test substance (rechallenge)
- Site: right flank (test substance) and left flank (70% ethanol) (challenge); right flank (rechallenge)
- Concentrations: 2% (challenge) and 0.2% (rechallenge)
- Evaluation: 24, 48 and 72 h after patch removal
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 2%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction intradermal: 1%; challenge: 2%
No. with + reactions:
16
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 2%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction intradermal: 1%; challenge: 2%
No. with + reactions:
15
Total no. in group:
20
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 2%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
Induction intradermal: 1%; challenge: 2%
No. with + reactions:
15
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 2%; rechallenge: 0.2%
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Induction intradermal: 1%; challenge: 2%; rechallenge: 0.2%
No. with + reactions:
0
Total no. in group:
20

Induction

Intradermal injections of Freund's complete adjuvant mixed with test substance or vehicle elicited irritation. A slight to moderate skin reaction was observed following the intradermal injections of the test substance. Moderate to severe skin reactions were observed at the backs of the test animals after the dermal induction.

 

Rechallenge

No positive skin reactions were registered indicating a possible dose-response relationship between challenge concentration and sensitization rate.

 

Body weight

The animals made normal body weight changes over the duration of the study and exhibited normal appearance and behaviour.

Interpretation of results:
other: Skin Sens. 1A
Remarks:
according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance revealed sensitising properties.
Executive summary:

A maximisation test in guinea pigs was performed according to OECD 406 and GLP in 1989.

The intradermal induction was 1 % in paraffin oil, topical induction was 25 % in EtOH 70 % and the challenge was performed with 2 % in EtOH 70 %. The rechallenge was 0.2 % in 70 % EtOH.

Challenge: 18/20 animals showed skin reactions at 24 hours, thereof 16 with grade >=2 and 15/20 had grade >=2 after 48 and 72 hours. In the control group 5/20 had grade 1 reactions after 24 hours, decreasing to 2 animals after 48 hours and 1 animal after 72 hours.

Rechallenge: In the test group and the control group only grade 1 reactions occurred.

According to this test the substance is a skin sensitiser (CLP Cat 1A).

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 Nov - 7 Dec 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No reliability check was reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1981
Deviations:
yes
Remarks:
no reliability check was reported
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was done before LLNA as first-choice method for in-vivo testing was set into force.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 310-370 g
- Housing: no data
- Diet: no data
- Water: no data
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 45-60
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal induction: arachis oill; dermal induction: absolute ethanol
Concentration / amount:
Induction: intradermal 0.5%, epicutaneous 10%
Day(s)/duration:
intradermal induction: on Day 0; dermal induction: on Day 7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: absolute ethanol
Concentration / amount:
1%
Day(s)/duration:
on Day 21; exposure period: 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (controls), 20 (test group)
Details on study design:
RANGE FINDING TESTS:
A range finding study was performed to determine the appropriate dose level of the test substance following intradermal and epicutaneous administrations. For the selection of the intradermal induction concentration test substance concentrations of 0.1, 0.5, 1 and 5% were investigated. A total of four animals were used, each animal receiving four injections of only one concentration of test substance. Animals were observed 24, 48 and 72 h and 7 days following treatment and any evidence of localized necrosis or systemic toxicity was recorded. Well-defined erythema was observed at 0.1 and 0.5% test substance concentration 24, 48 and 72 h after treatment and very slight erythema was observed after 7 days. Moderate erythema was observed 24 and 48 h and brown eschar was observed 72 h and 7 days after treatment with 1% test substance concentration. Beet red areas were observed 24 h, eschar formation 48 h and black eschar formation 72 h and 7 days after treatment with 5% test substance concentration. No evidence of systemic toxicity was noted. The concentration selected for the intradermal induction stage of the main study was 0.5%.
For the selection of the topical induction and challenge concentration eight animals (intradermal injected with FCA between 1 and 3 weeks earlier) were treated 24 h under occlusive conditions with test substance at a number of different concentrations (0.5, 1, 2, 5, 10, 25, 50 and 100%). Up to four patches were applied to each animal. Application sites were evaluated 1, 24 and 48 h after patch removal. No skin reactions were observed at 0.5 and 1% test substance concentration. Scattered mild redness (score 1) was observed in one animal and no skin reaction was observed in a second animal at 1% test substance concentration 24 h after patch removal. Moderate and diffuse redness (score 2) was observed at 5 and 10% test substance concentration 1, 24 and 48 h after patch removal. Intense redness and swelling (score 3) was noted at 25% test substance concentration 1, 24 and 48 h after patch removal. Furthermore, severe oedema over the whole flank was observed at 1 and 24 h at this concentration. Green coloured necrosis over the whole site surrounded by beet redness and severe oedema over the whole flank was noted at 50 and 100% test substance concentration. The concentration selected for the main study topical induction and challenge was 10% and 1%, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture FCA/water
Injection 2: 0.5% test substance in arachis oil
Injection 3: equal amounts of 0.5% test substance and FCA
Epicutaneous: 10% test substance in absolute ethanol
- Control group:
Injection 1: a 1:1 mixture FCA/water
Injection 2: arachis oil
Injection 3: equal amounts of 50% arachis oil and FCA
Epicutaneous: arachis oil
- Site: shoulder region (intradermal + epicutaneous)
- Frequency of applications: every 7 days
- Duration: Days 0-7
- Concentrations: intradermal 0.5%, epicutaneous 10%

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day of challenge: 21 (challenge)
- Exposure period: 24 h
- Test groups: 1% test substance in absolute ethanol
- Control group: 1% test substance in absolute ethanol
- Site: right flank (1% test substance) and left flank (absolute ethanol)
- Concentration: 1%
- Evaluation: 24 and 48 h after patch removal
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 1%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction intradermal: 0.5%; challenge: 10%
No. with + reactions:
20
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction intradermal: 0%; challenge: 1%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction intradermal: 0.5%; challenge: 1%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Desquamation was observed in 15/20 and fissuring in 4/20 animals.

Distinct signs of redness covering the whole flank were noted at the test substance sites of all experimental group animals at both the 24 and 48 h readings. Evidence of desquamation and fissuring were also noted after 48 h. No signs of redness were noted at the vehicle control sites of the experimental group animals or the test substance or vehicle control sites of the control group animals at either the 24 or 48-hour readings.

 

Body weight gains of animals in the experimental group between Day 0 and day 24 were comparable to those observed in the control group over the same period.

Interpretation of results:
other: Skin Sens. 1A
Remarks:
according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance revealed sensitising properties.
Executive summary:

A maximisation test in guinea pigs was performed according to OECD 406 and GLP in 1985.

The intradermal induction was 0.5 % in arachis oil, topical induction was 10 % in absolute EtOH and the challenge was performed with 1 % absolute EtOH.

Challenge: 20/20 animals showed skin reactions at 24 hours, thereof 18 with grade 2. After 48 hours desquamation was observed in 15/20 and fissuring in 4/20 of the treated animals.

In the control group no animal showed any reaction after 24 and 48 hours.

According to this test the substance is a skin sensitiser (CLP Cat 1A).

Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14 Jan - 21 Feb 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
No information on purity given and no reliability check performed.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The procedure used was the Open Epicutaneous Test as described by Klecak G, in Advances in Modern Toxicology Vol. 4 "Dermatotoxicology and Pharmacology" edited by Marzulli & Maibach pages 321 - 328.
GLP compliance:
yes
Type of study:
other: Klecak Open Epicutaneous Test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 300-373 g
- Housing: 4 animals per cage in solid-floor plastic cages, sawdust was used as bedding
- Diet: Guinea Pig FD1 Diet (Special Diet Services Limited, Witham, UK), ad libitum
- Water: mains tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 45-50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, open
Vehicle:
other: ethanol
Concentration / amount:
50 and 100%
Day(s)/duration:
100%: Day 0-11; 50%: Day 12-20
Adequacy of induction:
other: Due to severe skin reactions the test substance concentration was reduced on Day 12.
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: ethanol
Concentration / amount:
0.01, 0.1 and 1%
Day(s)/duration:
on Day 21
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, open
Vehicle:
other: ethanol
Concentration / amount:
0.01, 0.1 and 1%
Day(s)/duration:
on Day 35
Adequacy of challenge:
not specified
No. of animals per dose:
15
Details on study design:
RANGE FINDING TESTS:
No range finding test was conducted.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 21
- Exposure period: single epicutaneous
- Test group: test substance / test substance in absolute ethanol
- Control group: absolute ethanol
- Site: right flank (Day 0-5), unknown test site (Day 6-20) (test group); right flank (control group)
- Frequency of applications: daily
- Duration: Days 0-20
- Concentrations: 100% (Day 0-11), 50% (Day 12-20)

B. CHALLENGE EXPOSURE
- No. of exposures: 2 (challenge and rechallenge)
- Day of challenge: 21 (challenge) and 35 (rechallenge)
- Exposure period: single epicutaneous
- Test groups: 0.01, 0.1 and 1% test substance and absolute ethanol
- Control group: 0.01, 0.1 and 1% test substance and absolute ethanol
- Site: left flank
- Concentrations: 0.01, 0.1 and 1% (challenge and rechallenge)
- Evaluation: 24, 48 and 72 h after treatment
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Induction: 0%; challenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
Induction: 100% (Day 0-11), 50% (Day 12-20); challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15
Reading:
rechallenge
Hours after challenge:
72
Group:
negative control
Dose level:
Induction: 0%; challenge and rechallenge: 1%
No. with + reactions:
0
Total no. in group:
15

Table 1. Summary of results following challenge on Day 21 and Day 35.

Treatment group

Incidences of skin responses (score 1)

Day 22

Day 36

Challenge concentration

Challenge concentration

0.01%

0.1%

1%

V

0.01%

0.1%

1%

V

Treatment

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Control

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

V: vehicle

Body weight gains of animals in each of the experimental groups were comparable to those observed in the vehicle control group and were considered to be within normal limits.

Interpretation of results:
other: study cannot be used for classification on its own (non-OECD test protocol)
Conclusions:
Under the conditions of this Open Epicutaneous Test by Klecak the test substance revealed no sensitising properties.
Executive summary:

Klecak Open Epicutaneous Test in guinea pig with topical treatment undiluted from day 0 to 11, decreased to 50% in EtOH from day 12 to 20 due to strong irritation. Challenge with 0.01 %, 0.1 % and 1 % in EtOH.

No skin reactions in 15 treated and 15 control animals.

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

The skin sensitising potential of the test substance was investigated in a guinea pig maximisation test (GPMT test) performed according to OECD Guideline 406 and in compliance with GLP (1989). In this study the test substance revealed sensitising properties.

In a second GMPT test performed equivalent or similar to OECD Guideline 406 the test substance showed also sensitizing properties (1985).

In a third skin sensitisation test the test substance was investigated in an Open Epicutaneous Test by Klecak in guinea pig and revealed no sensitising properties (1986).

In a weight of evidence approach, the test substance is considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The skin sensitising potential of the test substance was investigated in a guinea pig maximisation test (GPMT test) performed according to OECD Guideline 406 and in compliance with GLP (1989). In this study the test substance revealed sensitising properties.

In a second GMPT test performed equivalent or similar to OECD Guideline 406 the test substance showed also sensitizing properties (1985).

In a third skin sensitisation test the test substance was investigated in an Open Epicutaneous Test by Klecak in guinea pig and revealed no sensitising properties (1986).

In a weight of evidence approach, the test substance is considered to be a skin sensitiser.

The available data on sensitisation of the test substance meet the criteria for classification as Skin Sens. 1A (H317) according to Regulation (EC) 1272/2008.