Climbazole

Endpoint summary

Description of key information

The results from three guideline-compliant studies (local lymph node assay, guinea pig maximisation test, guinea pig Buehler test) indicated that the substance did not exhibit any potential to induce skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

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.

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.

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.

Key result

Parameter:

SI

Value:

0.91

Test group / Remarks:

1%

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.08

Test group / Remarks:

20%

Key result

Parameter:

SI

Value:

7.77

Test group / Remarks:

positive control (HCA in AOO 35%)

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There were three studies (Symrise/Colgate Palmolive Company, 1979025; Bayer/Symrise, 1983063; and Symrise/P&G, 2007 Ref. 45) on guinea pigs and mice to evaluate the skin sensitization potential of climbazole (see below). The results from these studies confirm that climbazole did not exhibit any potential to induce skin sensitization in these animal models.

 

Magnusson Kligman guinea pig maximisation test (Symrise/Colgate Palmolive Company, 1979025):

In a skin sensitization study, guinea pigs were treated with Crinipan (BAY e 6975) according to the following schedule: Inductions on days 0 and 7: intradermal injections of a 10% formulation in 1,2-propanediol and Freund's Complete Adjuvant (FCA) and topical application (occlusive, 48 h) of a 10% formulation in 1,2-propanediol. Challenges on days 21 and 35 were made with epicutaneous application (occlusive, 24 h) of 10 and 1% or rather of 1 and 0.1% formulations in 1.2-propanediol. In each challenge test, 10 control animals were treated in the same manner as the animals in the test group. Treatment with crinipan did not affect the body weights of the animals, and no test agent-related deterioration of the animals' general condition was assumed by the author. Challenge with the 10% formulation caused skin reactions in animals of the test group as well as in the control animals. These reactions are considered a consequence of a primary irritation by crinipan, because the intensity and incidence (in all cases about 40-50%) in the two animal groups were comparable, and an irritating or sensitizing action of the formulation vehicle is not assumed by the author. After the challenge with less concentrated formulations, only animals pretreated with crinipan showed skin reddening. Three and two animals treated with the 1 and 0.1% formulation, respectively, showed positive skin reactions. These reactions are not considered a consequence of hypersensitivity in the sense of contact allergy, because the incidence of less than 20% was of a magnitude expected in view of the primary irritation effect noted (in the pilot test for irritation effect, one of the 4 animals showed positive skin findings after a single treatment with the 0.3% formulation). Since at the concentrations of crinipan and the vehicle used in this study, produced rather a irritation effects, the sensitization potential of crinipan could not be evaluated from these results, hence the results from this study are of supportive value.

 

Buehler test in guinea pigs (Bayer/Symrise, 1983063):

In an in vivo study in guinea pigs, crinipan (climbazole) did not cause skin sensitization when tested in a concentration range of 0.3 to 3% in a 3% aqueous solution of carboxymethyl cellulose (CMC). Female animals were induced with a 10% suspension of crinipan in 3% CMC by epicutaneous, occlusive application on the right shoulder-flank. A negative control group was treated with the vehicle (3% CMC) only and a positive control group was induced with 5 mL of a reference substance (diethyl ether containing 10 µg TGS) in parallel. This procedure was repeated once weekly for three weeks. After the third week, the guinea pigs were allowed to rest for one week and were then exposed to the challenge as follows: The test animals (induced with crinipan) and the negative control group were treated with three different concentrations 0.3, 1, and 3% of crinipan in 3% CMC, again by epicutaneous, occlusive application of 5 mL suspension to the shaven backs. Animals of the positive control group received 0.5 mL of the reference substance. After 24 h of exposure the challenge sites were depilated and after 3 h, evaluated for skin reactions. Skin inflammatory reactions occurring in the test and positive control group were compared to those in the negative control group. No reactions indicative of sensitization were observed in the animals challenged with 1 and 3% crinipan. In the group challenged with 0.3% crinipan, one of the 15 guinea pigs exhibited a slight erythema and oedema, which did not occur in the negative control group. However, since this reaction dissipated within 24 h and did not occur on challenge with higher concentrations, it is concluded that the reaction observed was not a sensitizing, but irritating effect. All animals of the positive control group (5/5) showed skin reactions (compared to the negative control), indicating a clear sensitizing effect of the reference substance and demonstrating that the guinea pigs used in this test were immunologically competent and thus, ensures the validity of the in vivo study. Based on the results of this skin sensitization test (Buehler test), crinipan is not considered to be a skin sensitizing agent at the concentrations up to 10%.

 

LLNA in mice (Symrise/P&G, 2007 Ref. 45):

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: acetone/olive oil (AOO; positive control vehicle); Group 7: 35% (w/v) HCA (positive control). On day 6, the mice were injected, i.v. with 20 µCi of 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 ß-scintillation counter to determine incorporation of the thymidine. All animals appeared normal throughout the study. The application sites 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 20% did not result in stimulation indices of 3 or greater and hence climbazole up to 20% exhibits no potential to induce dermal sensitization in LLNA.

 In these skin sensitization studies according to the above-mentioned methods/guidelines, crinipan did not cause dermal hypersensitivity reactions in the sense of contact allergy. Hence, crinipan is considered not to be a skin sensitizing agent under the described study conditions.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The respiratory sensitisation potential of crinipan was not studied. However, given the negative results in three skin sensitisation studies, it can be considered unlikely that the substance has any respiratory tract sensitisation potential.

Justification for classification or non-classification

In a guideline-compliant skin sensitisation studies (LLNA, maximisation and Buehler test in guinea pigs) the substance showed no skin sensitising properties. Therefore, the substance does not have to be classified as a skin sensitiser in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).