Registration Dossier

Administrative data

Description of key information

Weight of evidence: Skin sensitising, 2018

1. Skin sensitisation: sensitising (EC3 = 23.5%), female mice, eq. or similar to OECD TG 429, 2003

2. Skin sensitisation: non-sensitising, Guinea Pig (female), OECD TG 406, 2000

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:
key study
Study period:
03-02-2000 to 21-07-2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 1999 ; signature: December 1999
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.
Species:
guinea pig
Strain:
Hartley
Remarks:
Cri: (HA) BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 3 months old).
- Weight at study initiation: 362 ±16 g males and 343 ±15 g females
- Housing: Individually in polycarbonate cages (48 cm x 27 cm x 20 cm) with dust free sawdust and polypropylene water bottle.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): filtered (0.22 micron) water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 12 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 08-02-2000 to 13-03-2000
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
- Intradermal: 25% test material in Corn Oil
Day(s)/duration:
Day 1
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Concentration / amount:
- Topical: 100% test material (undiluted)
Day(s)/duration:
Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
Corn oil for the intradermal injections for the topical applications
Concentration / amount:
- Challenge: 100% (or in corn oil as applicable)
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
Corn oil for the intradermal injections for the topical applications
Concentration / amount:
- Challenge: 30% in corn oil
Day(s)/duration:
Day 32
No. of animals per dose:
Test group: 20; Control group: 10
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. By intradermal route: tested concentrations: 75%, 50% and 25% (w/w). Cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections. By cutaneous route (occlusive dressing, 24 hours) tested concentrations: 100% and 75% (w/w). Reactions were evaluated approximately 24 and 48 hours after removal. At 24 and 48 hours: intradermal route 25%w/w was selected due to slight irritation to irritation, and which remained at 6 days. In higher concentrations crust formation was observed at 6 days. At 24 and 48 hours: cutaneous route: no reactions were observed at 75% (w/w) or 100% concentration. Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 25% test material in Corn Oil
- Topical: 100% test material (undiluted)
- Challenge: 1st challenge: 100% (undiluted) ; 2nd challenge: 30%w/w in Corn Oil
Vehicle: Corn oil (intradermal induction and topical challenge), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test substance. The preparation of the test item at 0.9% NaCl solution (v/v) was non-homogenous with two phases, within in pre-testing solubility testing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in 0.9% NaCl; 2 ID: test item at 25% in corn oil ; 2 ID: a test mixture of the test item at 25% in FCA at 50% and in 0.9% NaCl equal volumes v/v.
- Control group: Vehicle and 50:50 FCA in 0.9% NaCl, and vehicle at 50% in 50:50 FCA/0.9% NaCl, only.
- Site: intradermal induction – three pairs of injections in clipped interscapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, clipped and SLS at 10% application. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) 1:1 w/w mixture of Freunds' Complete Adjuvant with 0.9% NaCl for injection; B) Test item at a 25% concentration ; C) A 1:1 w/w mixture of the test item, at twice the concentration used in (B) and Freunds' Complete Adjuvant/0.9% NaCl. Topical induction: The scapular area between the injection sites was clipped and brushed with a solution of SLS at 10%. Then the following day subsequently treated with 0.5 mL of a 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered along with injections of (A) and (C) in three sites.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 initial challenge ; 1 further challenge
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 22 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing. The second challenge was performed after a 9 day rest period.
- Test groups: 1 challenge; test item 100% and 2 challenge: 30%w/w in corn oil vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 100 and 30% using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24 and/or day 32).
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study
Challenge controls:
(Naive) negative control groups consisting of 5 females and 5 males were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole (1%w/w intradermal and 20%w/w topical)
Positive control results:
A reliability check was performed (presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Mercaptobenzothiazole (at 1%w/w intradermal induction and and 20%w/w topical concentrations) as positive control.
The skin reactions observed in seven experimental animals in response to the > 20 % PC item concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 80% to the 1%w/w induction and 20%w/w challenge concentrations.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 19.0. (one mortality at day 12) ; Clinical observations: None reported; maximum score = 1 (n=10) to 2 (n=3) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Remarks:
Were within the range of control group
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
19
Clinical observations:
Total no. in groups: 19.0. (one mortality at day 12) ; Clinical observations: None reported; maximum score = 1 (n=9) to 2 (n=1) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Remarks:
Were within the range of control group
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 1 (n=7) to 2 (n=1) (discrete or moderate erythema).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 1 (n=7) (discrete erythema).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1% intradermal induction and 20% cutaneous
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
non-concurrent PC conducted within 6 months of the definitive test; % sensitised was based on comparison of challenge sites with test and controls.
Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test item is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to guideline OECD TG 406 and EU Method B.6 and consistent with the Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 25%w/w in corn oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of ten males/females respectively and a control group of five males/females were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. Due to equivocal reactions a second challenge was conducted on day 32 with 30%w/w test item in corn oil. One mortality (male) was found on day 12 in the treatment group. No clinical signs were observed prior to mortality death. This was not attributed to. treatment with the test item. There were no clinical signs during the study. Bodyweight gain in the treated group was comparable to controls. In the first challenge, a discrete or moderate erythema (grade 1 or 2) was noted in 8/10 of the control group and in 13/19 of the treated group. Dryness of the skin was observed in one member of each group. In the second challenge, a discrete or moderate erythema (grade 1 or 2), sometimes together with dryness of the skin, was noted in 10/10 of the control group and in 13/19 of the treated group. As the cutaneous reactions observed after both challenge applications of the treated group were of similar incidence and severity when compared to those recorded in the control group, they were attributed to the irritant properties of the test item. Under the conditions of this study, the test item is not considered to be a contact skin sensitizer.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27-08-2003 to 21-11-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Method equivalent to guideline, well documented and performed under GLP. All relevant validity criteria were met.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: approximately 6 to 7 weeks old (prior to acclimatisation)
- Weight at study initiation: 17.3 – 24.8 grams
- Housing: Individually housed, in labelled shoebox style cages
- Diet: Free access to rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual: 19.4 to 23.4°C)
- Humidity (%): 40 to 80 (actual: 45 – 76%)
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 18-09-2003 To: 24-09-2003
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0% (vehicle control), 1%, 5%, 10%, 20% and 40%
ISOEUGENOL (positive control), 0.5%, 1.0% and 5.0%
No. of animals per dose:
Main test: 8 mice 0% (vehicle control) and 5 mice per test item dose group 1%, 5%, 10%, 20% and 40%
Details on study design:
RANGE FINDING TESTS:
Not applicable.

MAIN STUDY
- Compound solubility: Fully soluble in vehicle. The choice of AOO (4:1) as a vehicle was based on NIH Publication No. 99-449, "The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals / Compounds," in which AOO was the vehicle of choice.
- Irritation: None observed.
- Systemic toxicity: None observed. Three (3) of the 48 mice assigned to the study lost minimal amounts of weight between randomization and lymph node harvest (0.3 - 0.4 grams). The cause of this mild weight loss, distributed across treatment groups, is unknown.
- Ear thickness measurements: mean ear thickness values did not increase by 10% or more at any tested concentration of either isoeugenol (0.5% - 5.0%) or test item (1.0% - 40%).
- Erythema scores: No irritation was reported.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Five groups of five animals were treated with one test item concentration per group. One group of five animals was treated with vehicle.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day with monitoring for local and systemic toxicity during dosing. A rest period was allowed on days 4 and 5. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 2 μCi of 125-I labelled IuDR and 1x10^-5 M FuDR. After approximately five hours, all animals were terminated. Nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 20 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter.
- Tissue processing and radioactivity measurements: See above. Results from each cell suspension counted on the gamma counter were recorded in counts per minute (CPM). The CPM values were converted to disintegrations per minute (DPM) by dividing by the gamma counter efficiency and multiplying by 100. After the DPM values had been calculated, the mean "blank" DPM was subtracted from each individual DPM to obtain corrected DPM values. The mean corrected DPM and standard error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treatment-group mean DPM by the control (vehicle)-group mean DPM.

Observations:
- Mortality/Viability: Once daily
- Bodyweights: On Day 1 (pre-dose) and Day 6 (post termination).
- Clinical Observations: Once daily
- Irritation: Once daily.
- Ear Thickness: Ear measurements were taken prior to dosing on Days 1 and 3
Positive control substance(s):
other: Isoeugenol (CAS No 97-54-1)
Statistics:
A one-sample t test was performed to test whether the individual untransformed SI value for each dose level of each test/reference item was greater than or equal to 3. The natural log transformed DPM values for each compound were compared with vehicle using a Bartlett's Chi-Square test for variance homogeneity. If the Bartlett's Chi-Square results were found to be nonsignificant, a one-way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was found to be significant, then a Dunnett' s t test was performed using an alpha of 0.05. If the Barlett's Chi-Square was found to be significant, nonparametric analyses were conducted, specifically a Kruskal-Wallis (KW) test If the non-parametric analyses-were found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.

Calculations were performed using Microsoft® Excel and SAS®, version 8.
Positive control results:
The concurrent positive control (isoeugenol) had an EC3 = 1.55%. This was considered consistent with previously reported results.
Parameter:
EC3
Remarks:
%
Value:
23.5
Variability:
C.I. -%
Remarks on result:
other: See table below
Parameter:
SI
Value:
1
Variability:
± 0.1
Test group / Remarks:
1% in acetone:olive oil (4:1)
Parameter:
SI
Value:
1.3
Variability:
± 0.1
Test group / Remarks:
5% in acetone:olive oil (4:1)
Parameter:
SI
Value:
2.2
Variability:
± 0.3
Test group / Remarks:
20% in acetone:olive oil (4:1)
Parameter:
SI
Value:
7.9
Variability:
± 0.3
Test group / Remarks:
20% in acetone:olive oil (4:1)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The data per concentration was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The EC3 was determined from the appropriate regression equation.

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed.

BODY WEIGHTS: Three (3) of the 48 mice assigned to the study lost minimal amounts of weight between randomization and lymph node harvest (0.3 - 0.4 grams). The cause of this mild weight loss, distributed across treatment groups, is unknown. Applicant assessment indicates: the slight body weight would generally not be considered toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent or reported in the study.

Table 1. Results from the definitive test

Group

Number

CPM

DPM

Mean DPM

SD

SE

Mean SI

SE

Ears mean % increase

SE

Vehicle Control

1

18.8

21.3

 

 

 

 

 

 

 

 

2

14.8

16.2

 

 

 

 

 

 

 

 

3

46.4

56.2

 

 

 

 

 

 

 

 

4

19.8

22.5

 

 

 

 

 

 

 

 

5

21.6

24.8

 

 

 

 

 

 

 

 

6

21.8

25.1

 

 

 

 

 

 

 

 

7

24.2

28.1

 

 

 

 

 

 

 

 

8

23.0

26.6

27.6

12.1

4.3

-

-

2.1

1.49

Test Item 1%

9

20.0

22.8

 

 

 

 

 

 

 

 

10

21.2

24.3

 

 

 

 

 

 

 

 

11

31.2

37.0

 

 

 

 

 

 

 

 

12

24.2

28.1

 

 

 

 

 

 

 

 

13

23.6

27.3

27.9

5.5

2.5

1.0

0.1

3.0

1.05

Test Item 5%

14

22.0

25.3

 

 

 

 

 

 

 

 

15

28.6

33.7

 

 

 

 

 

 

 

 

16

25.8

30.1

 

 

 

 

 

 

 

 

17

16.2

18.0

 

 

 

 

 

 

 

 

18

35.4

42.3

29.9

9.1

4.1

1.1

0.1

3.5

1.15

Test Item 10%

19

25.2

29.4

 

 

 

 

 

 

 

 

20

33.4

39.7

 

 

 

 

 

 

 

 

21

34.4

41.0

 

 

 

 

 

 

 

 

22

31.4

37.2

 

 

 

 

 

 

 

 

23

31.0

36.7

36.8

4.5

2.0

1.3

0.1

2.4

0.99

Test Item 20%

24

68.4

84.1

 

 

 

 

 

 

 

 

25

36.6

43.8

 

 

 

 

 

 

 

 

26

42.6

51.4

 

 

 

 

 

 

 

 

27

54.0

65.8

 

 

 

 

 

 

 

 

28

46.8

56.7

60.4

15.5

6.9

2.2

0.3

3.9

1.10

Test Item 40%

29

164.2

205.3

 

 

 

 

 

 

 

 

30

162.6

203.3

 

 

 

 

 

 

 

 

31

184.6

231.1

 

 

 

 

 

 

 

 

32

227.4

285.3

 

 

 

 

 

 

 

 

33

130.4

162.5

217.5

45.2

20.2

7.9

0.7

3.1

0.25

Isoeugenol 0.5%

34

43.2

52.2

 

 

 

 

 

 

 

 

35

29.4

34.7

 

 

 

 

 

 

 

 

36

48.4

58.7

 

 

 

 

 

 

 

 

37

37.0

44.3

 

 

 

 

 

 

 

 

38

29.0

34.2

44.8

10.8

4.8

1.6

0.2

3.1

1.14

Isoeugenol 1.0%

39

71.8

88.4

 

 

 

 

 

 

 

 

40

45.8

55.4

 

 

 

 

 

 

 

 

41

64.6

79.2

 

 

 

 

 

 

 

 

42

41.4

49.9

 

 

 

 

 

 

 

 

43

27.4

32.2

61.0

22.7

10.2

2.2

0.4

4.74

0.62

Isoeugenol 5.0%

44

82.4

101.8

 

 

 

 

 

 

 

 

45

162.4

203.0

 

 

 

 

 

 

 

 

46

231.8

290.9

 

 

 

 

 

 

 

 

47

104.8

130.1

 

 

 

 

 

 

 

 

48

251.0

315.2

208.2

94.5

42.3

7.5

1.5

5.6

1.46

 

 

 

 

 

 

 

 

 

 

 

Where:

SE = standard error of the mean

SD = standard deviation

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test item is considered to be sensitising to skin with EC3 of 23.5%.
Executive summary:

The study was performed to using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of: 1%, 5%, 10%, 20% and 40% in acetone/olive oil (4:1) and with a 0% vehicle control group. A concurrent positive control consisting of Isoeugenol at 0.5%, 1.0% and 5.0% concentrations was additionally conducted. Observations were made daily and ear thickness measurements conducted daily on days 1 to 3. No irritation or signs of systemic toxicity were observed. Three days after the final auricular application, the animals were injected intravenously with 125-I radiolabelled Iododeoxyuridine to label proliferating cells. 125-I incorporation was quantified using a gamma counter. The SI values calculated for the test item concentrations 1%, 5%, 10%, 20% and 40% were 1.0, 1.1, 1.3, 2.2 and 7.9 respectively. The results show that the test item elicited an SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 23.5%. The concurrent positive control Isoeugenol had an EC3 value of 1.55%. Under the conditions of this study, the test item would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.

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

Key study: in vivo, eq. or similar to OECD TG 429, 2003 : The study was performed to using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of: 1%, 5%, 10%, 20% and 40% in acetone/olive oil (4:1) and with a 0% vehicle control group. A concurrent positive control consisting of Isoeugenol at 0.5%, 1.0% and 5.0% concentrations was additionally conducted. Observations were made daily and ear thickness measurements conducted daily on days 1 to 3. No irritation or signs of systemic toxicity were observed. Three days after the final auricular application, the animals were injected intravenously with 125-I radiolabelled Iododeoxyuridine to label proliferating cells. 125-I incorporation was quantified using a gamma counter. The SI values calculated for the test item concentrations 1%, 5%, 10%, 20% and 40% were 1.0, 1.1, 1.3, 2.2 and 7.9 respectively. The results show that the test item elicited an SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 23.5%. The concurrent positive control Isoeugenol had an EC3 value of 1.55%. Under the conditions of this study, the test item would be considered to be classified under Regulation (EC) No 1272/2008 as skin sensitizer category 1B.

Key study: in vivo, OECD TG 406, 2000 : The study was performed according to guideline OECD TG 406 and EU Method B.6 and consistent with the Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 25%w/w in corn oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of ten males/females respectively and a control group of five males/females were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. Due to equivocal reactions a second challenge was conducted on day 32 with 30%w/w test item in corn oil. One mortality (male) was found on day 12 in the treatment group. No clinical signs were observed prior to mortality death. This was not attributed to. treatment with the test item. There were no clinical signs during the study. Bodyweight gain in the treated group was comparable to controls. In the first challenge, a discrete or moderate erythema (grade 1 or 2) was noted in 8/10 of the control group and in 13/19 of the treated group. Dryness of the skin was observed in one member of each group. In the second challenge, a discrete or moderate erythema (grade 1 or 2), sometimes together with dryness of the skin, was noted in 10/10 of the control group and in 13/19 of the treated group. As the cutaneous reactions observed after both challenge applications of the treated group were of similar incidence and severity when compared to those recorded in the control group, they were attributed to the irritant properties of the test item. Under the conditions of this study, the test item is not considered to be a contact skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B.

 

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.