Climbazole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-08- 21 to 2007-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

Additionally, the study is in line with the recommendations of the Scientific Committee on Cosmetic and Non-Food Products intended for consumers as laid down in the SCCNFP's Note of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation, 5th Revision, adopted by the SCCNFP during the 25th plenary meeting of 20 October 2003, SCCNFP/0690/03 Final.

GLP compliance:

yes

Remarks:

Exception: The test article was caracterized for composition, purity and stability. Certificates of Analysis were provided. However, the analyses were not performed under GLP/GMP.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Climbazole

EC Number:

253-775-4

EC Name:

Climbazole

Cas Number:

38083-17-9

Molecular formula:

C15H17ClN2O2

IUPAC Name:

1-(4-chlorophenoxy)-1-(1H-imidazol-1-yl)-3,3-dimethylbutan-2-one

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

Female mice of 8-9 weeks of age and with 18-23 g of body weight were included in the study.
Housing: Animals were group-housed (5 per cage) upon receipt then individually housed prior to the initiation of dosing. No other species were kept in the same room.
Lighting: 12 h light/12 h dark; Temperature: 23.3 to 29.4°C; Relative humidity: 31 to 52%; Food: Animals had access to Certified Rodent Chow ad libitum; Water: Tap water was available ad libitum, via water bottles; Acclimatisation: Study animals were acclimated to their housing for 5 days prior to their first day of dosing.

Study design: in vivo (LLNA)

Vehicle:

other: Vehicle for test item: DMSO. Vehicle for positive control: acetone/olive oil, 4:1 [AOO]

Concentration:

7 groups of 5 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 days with the test article (climbazole) at 1, 5, 10, 20% (w/v) dissolved in the vehicle DMSO, with the vehicle for the positive control AOO, or with the positive control (HCA at 35%).

No. of animals per dose:

5 female mice

Details on study design:

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
On each day of dosing, the test article was prepared at the appropriate concentrations (w/v) in volumetric flasks by dissolving the appropriate amount of test article in DMSO. All preparations were vortexed to mix. Dosing formulations were clear-colorless to off-white liquids.

On each day of dosing, the positive control Α-Hexylcinnamaldehyde (HCA) was prepared at 35% in a glass tube by dissolving the appropriate volume of HCA in Acetone:olive oil:: 4:1 (AOO). The preparation was vortexed to mix and dosed as a clear yellow solution.

7 groups of 5 CBA/J female mice were treated on the dorsal surface of both ears once per day for 3 consecutive days as follows:

GROUP TREATMENT VEHICLE DOSE
1 Vehicle DMSO -
2 Climbazole (GTS20788) DMSO 1% (w/v)
3 Climbazole (GTS20788) DMSO 5% (w/v)
4 Climbazole (GTS20788) DMSO 10% (w/v)
5 Climbazole (GTS20788) DMSO 20% (w/v)
6 Positive Control Vehicle AOO -
7 HCA AOO 35% (w/v)

On day 6, the mice were injected, i.v., with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were treated with 5% trichloroacetic acid (TCA) to precipitate the DNA. The resulting pellets were counted in a β-scintillation counter to determine incorporation of the 3H-thymidine.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis of the DPM data was performed using SAS version 6.12, a statistical analysis software package developed by SAS institute Inc. Individual DPM values were analyzed by log transformation (base 10) of the data. Comparisons with the control group were based on the least significante difference criterion. All statistical tests were conducted at a 1%, two-tailed probability level.

Body weight and change in body weight means and SEM were calculated for each group. Evaluation of equality of means were made by a one way analysis of variance using the F distribution to assess statistical significance using SYSTAT version 9.01 developed by SPSS, Inc. If statistically significant differences were found, a Dunnett’s test was used to determine the degree of significance from the control mean.

Results and discussion

Positive control results:

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.77 (see Table below). A 3-fold or greater increase in proliferative activity relative to the concurrent AOO control is considered a positive response.

Table: Results
Group Treatment Dose DPM(mean ± sem) SI (Test/control Ratio) Results*
1 DMSO - 631 ± 69 - -
2 Climbazole 1% (w/v) 573 ± 91 0.91 -
3 Climbazole 5% (w/v) 480 ± 86 0.76 -
4 Climbazole 10% (w/v) 751 ± 138 1.19 -
5 Climbazole 20% (w/v) 684 ± 56 1.08 -
6 Acetone:olive oil 4:1(AOO) - 590 ± 87 - -
7 HCA in AOO 35% (w/v) 4586 ± 855** 7.77 +

*Test/control ratio of 3 or greater represents a positive result
**Statistically significant difference when Log DPM compared to the corresponding vehicle control group (p<0.001)

All animals appeared normal throughout the study. The application sites on the mice from the positive control group appeared wet on days 2-5.

Mean body weights at day 1 and day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test articles did not appear to cause any overt toxicity.

At termination, the lymph nodes of the mice treated with the positive control were enlarged relative to the lymph nodes from the positive control vehicle treated mice but otherwise appeared normal.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

1%

Results 2

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

10%

Results 4

Key result

Parameter:

SI

Value:

1.08

Test group / Remarks:

20%

Results 5

Key result

Parameter:

SI

Value:

7.77

Test group / Remarks:

positive control (HCA in AOO 35%)

Any other information on results incl. tables

Dosing solutions were analyzed for concentration. All, day 1 dose formulations met the acceptance criteria of 90 -110% of target concentration and ≤5% RSD and were determined to be accurately prepared. No test article was detected in the vehicle control sample.

Samples from the top, middle and bottom of the 1 and 20% dose formulations were also analyzed to assess the homogeneity and stability. All samples met the acceptance criteria and were determined to be homogeneous and stable at 2-8°C for at least eight weeks.

Mean body weights at day 1 and day 6 and mean changes in body weights were evaluated (Table below). There were no statistically significant differences observed between any of the treatment groups. Therefore, the test articles did not appear to cause any overt toxicity.

Table: Body weights and body weight change

Group	Treatment	Dose (%)	Body Weights (g) (mean± sem)		Change in Body Weight (g) (mean± sem)
			Day 1	Day 6
1	DMSO	-	19.4±0.5	21.0±0.7	1.6±0.5
2	Climbazole	1% (w/v)	20.2±0.5	21.2±0.7	1.0±0.4
3	Climbazole	5% (w/v)	20.2±0.4	20.0±0.4	-0.2±0.2
4	Climbazole	10% (w/v)	20.0±0.5	20.2±0.4	0.2±0.5
5	Climbazole	20% (w/v)	20.0±0.5	20.2±0.4	0.2±0.5
6	Acetone:olive oil 4:1 (AOO)	-	20.6±0.5	21.4±0.5	0.8±0.4
7	HCA in AOO	35% (w/v)	20.4±0.7	20.4±0.7	0.0±0.3

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information up to 20% (w/v) of climbazole tested

Conclusions:

A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3 -fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index ≥3 is regarded as a positive response. Treatment with climbazole at doses up to and including 20% did not result in stimulation indices of 3 or greater and hance is not considered to have skin sensitizing activity.

Executive summary:

The purpose of this study was to determine if the test articles would induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes.

Seven groups of 5 CBA/J female mice each were treated on the dorsal surface of both ears once per day for 3 consecutive days. Group 1: DMSO (vehicle control); Group 2: 1% (w/v) climbazole; Group 3: 5% (w/v) climbazole; Group 4: 10% (w/v) climbazole; Group 5: 20% (w/v) climbazole; Group 6: AOO (positive control vehicle); Group 7: 35% (w/v) HCA.

 

On day 6, the mice were injected, i.v. with 20 µCi of 3H-thymidine in sterile saline. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were treated with 5% TCA to precipitate the DNA. The resulting pellets were counted in a ß-scintillation counter to determine incorporation of the 3H-thymidine.

All animals appeared normal throughout the study. The application sites on the mice from the positive control group appeared wet on days 2-5. The AOO treated animals exhibited wet ears on day 3-5. Mild signs of skin irritation in the form of very slight edema were noted on the ears of the animals treated with 1% climbazole on day 4, and on the ears of the animals treated with 5, 10 and 20% climbazole on days 3 and 4.

Mean body weights at day 1 and day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity. At termination, the lymph nodes of the mice treated with the positive control were enlarged relative to the lymph nodes from the positive control vehicle treated mice but otherwise appeared normal. The lymph nodes from the mice in the vehicle treated groups and all test article treated groups were normal in size and appearance.

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 7.77. A 3-fold or greater increase in proliferative activity relative to the concurrent AOO control is considered a positive response. Thus, the sensitivity of the test system was demonstrated.

Exposure to climbazole at 1, 5, 10 and 20% (w/v) resulted in stimulation indices of 0.91, 0.76, 1.19, and 1.08, respectively. No statistically significant differences were found between the groups treated with climbazole and the DMSO vehicle control. Treatment with climbazole at doses up to and including 20% did not result in stimulation indices of 3 or greater and hence is not considered to have skin sensitizing activity.