Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 406): sensitising

Skin sensitisation (OECD 406): not sensitising

Skin sensitisation (OECD 429): sensitising

WoE conclusion from in vivo skin sensitisation tests: sensitising

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:
12 Nov - 05 Dec 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical purity of test substance given.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
yes
Remarks:
No purity of the test substance given.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission directive 92/69/EEC, 1992
Deviations:
not specified
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
- Source:Mollegaard Breeding and Research Centre A/S, Ejby, DK-4623 Lille Skensved
- Females nulliparous and non-pregnant: [not specified]
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 315 - 343 g
- Housing: two to three animals per cage, in polycarbonate (macrolone type IV, floor area 1800cm²) cages, softwood sawdust bedding
- Diet: Altromin 3113 (Chr. Petersen A/S, Ringsted, Denmark), ad libitum
- Water: vitamin C enriched domestic quality water acidified to pH 2.5 with hydrochloric acid
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
other: sesame oil and a 1:1 (w/w) mixture of Freund´s complete adjuvant and sesame oil
Concentration / amount:
10% (w/w)
Day(s)/duration:
single injection
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25%
Day(s)/duration:
48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25% (w/w)
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 (control), 20 (test group)
Details on study design:
RANGE FINDING TESTS:
The aim of the range finding test was to identify the highest concentrations which caused mild-to-moderate skin irritation after intracutaneous and epicutaneous administration and the highest non-irritant concentration after epicutaneous administration. For the intracutaneous irritancy test two animals were treated with 4 concentrations of the test substance (1.25, 2.5, 5 and 10%) in ethanol/diethylphthalate 1:1 (w/w). All concentrations tested induced erythema (grade 1) 24 and 48 h after administration and 10% was selected as adequate concentration for the intradermal induction phase of the main study. For the epicutaneous irritancy test the clipped/shaved flanks of two animals were treated with test substance concentrations of 6.25, 12.5 and 25% (w/w) under occlusive dressings for 24 and 48 h. The test substance did not cause skin reactions at any concentration tested, therefore 25% (w/w) was chosen as adequate concentration for epicutaneous induction and challenge application of the main study.

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 (v/v) FCA/water
Injection 2: 10% (w/w) test substance in sesame oil
Injection 3: 10% (w/w) test substance in a 1:1 mixture (w/w) FCA/sesame oil
Epicutaneous: 25% (w/w) test substance in a 1:1 mixture (w/w) ethanol/diethylphthalate

- Control groups:
Intradermal (3 pair of injections):
Injection 1: a 1:1 mixture (w/w) FCA/water
Injection 2: sesame oil
Injection 3: a 1:1 mixture (w/w) FCA/water
Epicutaneous: sesame oil

- Site: intradermal: shoulder region (intradermal + epicutaneous: cranial: injection 1 and 2; caudal: injection 3)
- Frequency of applications: every 7 days
- Duration: Day 0-9
- Concentrations: intradermal: 10%; epicutaneous: 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 20 - 21
- Exposure period: 24 h
- Test groups: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Control group: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Site: left anterior flank (test substance) and left posterior flank (vehicle)
- Concentration: 25%
- Evaluation (hr after challenge): 24 and 48 h

OTHER:
To provoke a mild irritation, pretreatment of the clipped skin with 10% sodium lauryl sulphate in petrolatum was carried out the day before induction by epicutaneous administration.

Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
Formaldehyde: 0.1% (intradermal) and 5% (epicutaneous induction and challenge) in distilled water (w/w)
Positive control results:
The positive control is a historical background data group from a study performed during 10 Oct - 07 Nov 1996 under same experimental conditions. In this study 90% of the animals responded positively.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 10% Challenge: 0%
No. with + reactions:
2
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 10% Challenge: 25%
No. with + reactions:
12
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenege: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 10% Challenge: 0%
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 10% Challenge: 25%
No. with + reactions:
13
Total no. in group:
20

Table1. Individual irritation scores 24 and 48 h after challenge

Animal No.

24 h after challenge

48 h after challenge

Left anterior (25%)

Left posterior (vehicle)

Left anterior (25%)

Left posterior (vehicle)

Control group

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

0

0

5

0

0

0

0

6

0

0

0

0

7

0

0

0

0

8

0

0

0

0

9

0

0

0

0

10

0

0

0

0

Test group

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

1

0

5

0

0

0

0

6

0

0

0

0

7

2

1

2

1

8

0

0

0

0

9

2

0

1

0

10

1

0

0

0

11

2

0

2

0

12

2

0

2

0

13

2

0

3

0

14

1

0

2

0

15

0

0

2

0

16

2

0

2

0

17

2

0

1

0

18

2

0

1

0

19

2

0

3

0

20

2

1

2

0

0: no visible change

1: slight or discrete erythema

2: moderate and confluent erythema

3: intense erythema and swelling

Intradermal injections of Freund' s complete adjuvant mixed with vehicle or test substance elicited irritation. No skin reactions were observed following induction with either vehicle or test substance.

No animal died or showed clinical signs during the course of investigation and the mean value for body weight and the body weight gain were not affected by the treatment.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance produced mild to intense (grade 1-3) erythema in 12/40 animals 24 h after challenge and in 13/40 animals 48 h after challenge. Therefore, the test substance is considered to be skin sensitising.
Executive summary:

The dermal sensitizing potential was investigated according to OECD Guidelines No. 406, 1992. Thirty animals divided into a test group of 20 animals and a negative control group of 10 animals were included in the study. The study comprised an induction and a challenge phase. The animals in the test group were induced with the test article whereas the animals in the control group were induced with sesame oil or Ethanol/diethylphalate 1:1. The induction procedure included intradermal injections and a closed patch topical application one week apart. A 10% (w/w) test article concentration in sesame oil was used for the intradermal induction. A 25% (w/w) test article concentration in Ethanol/Diethylphthalate 1:1 was used for the topical

induction and for the challenge application. Under these experimental conditions evidence of delayed contact hypersensitivity was seen in 12 out of 20 animals. The substance is considered to be a sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 May - 11 Jun 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical purity of the test substance given; no skin reactions at 25% after epicutaneous induction; no treatment with SLS; concentration may be to low
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
yes
Remarks:
No analytical purity of the test substance given; no skin reactions at 25% after epicutaneous induction; no treatment with SLS; concentration may be too low
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission directive No 92/69/EEC, 1992
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
National Board of Health, Bronshoj, Denmark
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
- Source: Mollegaard Breeding and Research Centre A/S,Lille Skensved, Denmark
- Females nulliparous and non-pregnant: [not specified]
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 287- 335 g
- Housing: two to three animals per cage, in polycarbonate (macrolone type IV, floor area 1800cm²) cages, softwood sawdust bedding
- Diet: Altromin 3113 (Chr. Petersen A/S, Ringsted, Denmark), ad libitum
- Water: vitamin C enriched domestic quality water acidified to pH 2.5 with hydrochloric acid
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): (12 / 12)
Route:
intradermal
Vehicle:
other: sesame oil and a 1:1 (w/w) mixture of FCA and saline
Concentration / amount:
1.25%
Day(s)/duration:
single injection
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25%
Day(s)/duration:
48 h
Adequacy of induction:
other: concentration non-irritant, no pre-treatment with SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25%
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
25%
Day(s)/duration:
24 h
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/diethylphthalate 1:1
Concentration / amount:
12.5%
Day(s)/duration:
24 h
Adequacy of challenge:
not specified
No. of animals per dose:
10 (control), 20 (test group)
Details on study design:
RANGE FINDING TESTS:
The aim of the range finding test was to find out the highest concentrations which cause mild-to-moderate skin irritation after intracutaneous and epicutaneous administration and the highest non-irritant concentration after epicutaneous administration. For the intracutaneous irritancy test two animals were treated with 3 concentrations of the test substance (1.25, 2.5 and 5%) in ethanol/diethylphthalate 1:1 (w/w). All concentrations tested induced erythema (grade 1) 24 and 48 h after administration and 1.25% was selected for the intradermal induction phase of the main study. For the epicutaneous irritany test the clipped flanks of two animals were treated with test substance concentrations of 3.12, 6.25, 12.5 and 25% (w/w) under occlusive dressings for 24 and 48 h. The test substance did not cause skin reactions at any concentration tested, therefore the 25% (w/w) concentration was chosen for epicutaneous induction and challenge and 25 and 12.5% was chosen for re-challenge application of the main study.

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 (w/w) FCA/saline
Injection 2: 1.25% (w/w) test substance in sesame oil
Injection 3: 1.25% (w/w) test substance in a 1:1 mixture (w/w) FCA/saline
Epicutaneous: 25% (w/w) test substance in a 1:1 mixture (w/w) ethanol/diethylphthalate

- Control groups:
Intradermal (3 pair of injections):
Injection 1: a 1:1 mixture (w/w) FCA/saline
Injection 2: sesame oil
Injection 3: a 1:1 mixture (w/w) FCA/saline
Epicutaneous: 1:1 mixture (w/w) ethanol/diethylphthalate

- Site: intradermal: shoulder region (intradermal + epicutaneous: cranial: injection 1 and 2; caudal: injection 3)
- Frequency of applications: every 7 days
- Duration: Day 1-10
- Concentrations: intradermal: 1.25%; epicutaneous: 25%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: Day 21 - 22 and Day 35 - 36
- Exposure period: 24 h
- Test groups: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Control group: test substance in vehicle (ethanol/diethylphthalate 1:1) and vehicle only
- Site: left anterior flank (test substance) and left posterior flank (vehicle)
- Concentrations: 25 (challenge # 1 and rechallenge) and 12.5% (rechallenge)
- Evaluation (hr after challenge): 24 and 48 h

OTHER:
No SDS pre-treatment, although test substance was non-irritating at 25%.
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde: 1.25% (intradermal) and undiluted (epicutaneous)
Positive control results:
The positive control is a historical background data group from a study performed during 30 Dec 1997 - 29 Jan 1998 under same experimental conditions. 9/10 and 7/10 animals showed positive skin reactions (grade 1-2) 24 h and 48 h after challenge, respectively.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
1
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 1.25% Challenge: 0%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 1.25% Challenge: 25%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Challenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 1.25% Challenge: 0%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 1.25% Challenge: 25%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 12.5%
No. with + reactions:
1
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 0%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 12.5%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 25%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 12.5%
No. with + reactions:
1
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0% Rechallenge: 25%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 0%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 12.5%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 1.25% Rechallenge: 25%
No. with + reactions:
0
Total no. in group:
20

Intradermal injections of Freund' s complete adjuvant mixed with vehicle or test substance elicited irritation. On Day 3 slight erythema was observed at the test substance injection site. No skin reactions were observed following induction with the vehicle.

No animal died or showed clinical signs during the course of investigation and the mean value for body weight and the body weight gain were not affected by the treatment.

Challenge: In the control group discrete or patchy erythema (grade 1) was observed in 1/10 animals 24 h after challenge with the test substance solution of 25%. No skin reactions were observed in any animal and any test substance concentration 24 or 48 h after first challenge.

Rechallenge:

In the control group discrete or patchy erythema (grade 1) was observed in 1/10 animals 24 after rechallenge with the 25% solution of the test substance and in 1/10 animals 24 and 48 h after rechallenge with the 12.5% solution of the test substance. No skin reactions were observed in any animal of the test group at any test substance concentration 24 or 48 h after rechallenge.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the guinea pig maximisation test the test substance revealed no sensitising properties.
Executive summary:

The dermal sensitising potential was investigated according to OECD Guideline 406, 1992. Thirty animals divided into a test group of 20 animals and a negative control group of 10 animals were included in the study. The study comprised an induction and a challenge phase. The animals in the test group were induced with the test article on sesame oil for intradermal induction and test article in Ethanol/diethyl phthalate 1:1 for dermal induction. The animals in the control group were induced with sesame oil for intradermal induction and Ethanol/diethyl phthalate 1:1 for dermal induction. The induction procedure included intradermal injections and a closed patch topical application one week apart.

The skin reactions were evaluated 24 and 48 hours after termination of the challenge procedure and 24 and 48 hours after termination of the rechallenge procedure. A 1.25% (w/w) test article concentration in sesame oil was used for the intradermal induction. A 25% (w/w) test article concentration in Ethanol/diethyl phthalate 1:1 was used for the topical induction and a 25% (w/w) test article concentration in Ethanol/diethyl phthalate 1:1 was used for the challenge application. For the rechallenge application 25% or 12.5% (w/w) test article concentrations in Ethanol/diethyl phthalate 1:1 were used.

Under these experimental conditions no evidence of delayed contact hypersensitivity was seen.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 - 16 Jun 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
no rationale for dose selection; no pre-screen test; no body weight measurement
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
pre-guideline
Deviations:
yes
Remarks:
no rationale for dose selection; no pre-screen test; no body weight measurement
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca/Ola/hsd
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Bicester, UK
- Housing: maximum of 4 mice per cage
- Diet: R&M No. 1 (Special Diet Services Limited, Witham, UK), ad libitum
- Water: mains water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: To: 11 - 16 Jun 1997
Vehicle:
other: acetone
Concentration:
Test substacne: 1, 10 and 30%
Positive control: 1, 3 and 10%
No. of animals per dose:
4
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The criterion for a positve response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance or vehicle on Day 1. The application was repeated on Day 2 and 3. Three days after the third application an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each individual animal. A single cell suspension was prepared by gentle separation through a 200 mesh stainless steel gauze. The cell suspensions were washed three times with an excess of PBS and precipitated with 5% trichloroacetic acid at 4°C overnight. The pellets were resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid prior to ß-scintillation.

POSITIVE CONTROL STUDY: A positive control study was conducted seperately from main study using hexylcinnamaldehyde to ensure that the test system continues to respond to a known sensitising chemical.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
SI values for positive control were 2.48, 5.35 and 7.96 at 1, 3 and 10%, respectively.
Key result
Parameter:
SI
Value:
2.06
Test group / Remarks:
1% (w/v)
Key result
Parameter:
SI
Value:
5.77
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
5.87
Test group / Remarks:
30% (w/v)
Key result
Parameter:
EC3
Value:
3.28

Table 1. Results of LLNA

Concentration of test substance (% w/v)

Number of lymph nodes assayed

Counts per minute (cpm)

cpm per lymph node (x10 -² )

SI (test: control ratio)

0 (vehicle)

8

423

0.53

N/A

1

8

873

1.09

2.06

10

6*

1836

3.06

5.77

30

8

2489

3.11

5.87

Positive control (hexylcinnamaldehyd)

0 (vehicle)

8

929

1.16

N/A

1

8

2300

2.88

2.48

3

8

4964

6.21

5.35

10

8

7382

9.23

7.96

* one animal was found dead on Day 5; death not considered to be test substance related

N/A : not applicable

CLINICAL OBSERVATIONS:

One animal was found dead during the study (Day 5). However, in the absence of any signs of systemic toxicity at any dose level the death is considered not to be related to administration of the test substance.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the mouse Local Lymph Node Assay SI values were 2.06, 5.77 and 5.87 at test substance concentrations of 1, 10 and 30%. The EC3 value was 3.28. Thus, under conditions of this test the test substance is considered to be skin sensitising.
Executive summary:

Test item was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay similar to OECD 429. The test substance was applied as 1%, 10% or 30% w/v preparations in acetone. The test substance was shown to have the capacity to cause skin sensitisation when applied as 10% or 30% w/v preparations in acetone. Hexylcirmamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 3 or 10% w/v preparations in acetone, confirming the validity of the protocol used for this study. In conclusion, test item is a skin sensitiser under the conditions of the test.

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

Two in vivo GPMT and one LLNA is available for the test substance.

The skin sensitising potential of the test substance was investigated in vivo by a guinea pig maximisation test (GPMT) according to OECD Guideline 406 in compliance with GLP (1996). After intradermal induction with a 10% solution of the test substance in sesame oil, skin was pretreated with 10% SDS and epicutaneous induction with a 25% solution of the test substance in ethanol/diethylphthalate 1:1 was applied by occlusive patches. Animals were challenged epicutaneously with 25% solution of the test substance in ethanol/diethylphthalate 1:1. No skin reactions were observed following induction. Slight to moderate erythema (grade 1 - 2) was observed in 12/20 animals 24 h after challenge and slight to intense erythema (grade 1 - 3) was observed in 13/20 animals 48 h after challenge with the test substance. Based on the results of this GPMT, the test substance was regarded as a skin sensitizer.

In a second guinea pig maximisation test (GPMT) according to OECD Guideline 406 in compliance with GLP (1998) induction was conducted by intradermal application of 1.25% and conducted by intradermal application of 25% solutions of the test substance in sesame oil and ethanol/diethylphthalate 1:1, respectively. On Day 3 slight erythema was observed at the test substance injection site. Animals were challenged epicutaneously with a 25% solution of the test substance in ethanol/diethylphthalate 1:1 and rechallenged with a 25 and 12.5% solution of the test substance in ethanol/diethylphthalate 1:1. No skin reactions were observed in any animal 24 and 48 h after challenge and rechallenge with both test substance concentrations. Based on the results of this GPMT, the test substance is not considered to be skin sensitizing.

In a third in vivo test, the skin sensitising potential of the test substance was evaluated in a Local Lymph Node Assay (LLNA) similar to OECD Guideline 429 and in compliance with GLP (1997). Treatment of mice with the test substance revealed stimulation indices of 2.06, 5.77 and 5.87 at test substance concentrations of 1, 10 and 30% in acetone, respectively. The EC3 was calculated to be 3.28%. In conclusion, based on the available data the test substance is considered to be a weak skin sensitiser.

Conclusion:

Based on one negative and two positive in vivo skin sensitisation tests the test substance is considered to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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