Climbazole

Endpoint summary

Description of key information

The skin and eye irritation properties of crinipan were evaluated in an in vitro study in the human skin model EpiDerm, in a 3-week in vivo skin irritation study in rabbits and a series of 4 skin irritation patch test in human volunteers.
The eye irritation properties of crinipan were studied in two in vitro models, the HET-CAM and the BCOP test, as well as in an in vivo study in rabbits.
No effects indicative of any eye or skin irritation potential were observed. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-12 to 2010-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to EU-Method B.46 " In vitro Skin Irritation: Reconstructed human epidermis model test" (23. July 2009)

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: According to Human Skin Model Test EU-Method B.46 adopted 23. July 2009

Deviations:

no

Principles of method if other than guideline:

The in vitro skin irritation test is used to determine the skin irritancy of chemicals by measuring the cell viability in a reconstructed human epidermis (RhE) model after applying the test substance. The RhE model consists of human-derived epidermal keratinocytes, which have been cultured to form a multilayered model of the human epidermis. Irritant chemicals are identified by their ability to decrease cell viability by quantitatively measuring the enzymatic conversion of the vital dye MTT (3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide) to a blue formazan salt. Skin irritation potential of the test substance is assessed by reducing the formazan production to less than 50% of the negative control.

GLP compliance:

yes (incl. QA statement)

Species:

other: not applicable (this is an in vitro test)

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

other: not applicable (this is an in vitro test)

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable (this is an in vitro test)

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Amount of test item: mean: 22.7 mg

Duration of treatment / exposure:

60 min

Observation period:

not applicable (cell viability was determined after the end of treatment period)

Number of animals:

not applicable (this is an in vitro test)

Details on study design:

The human skin model test was performed with commercially available Epi-200-SIT-Kit. The EpiDermTM tissues were produced from MatTek Corporation in Ashland, USA. The day of delivery was the 01. July 2010 batch: 13657

Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) without CaCl2 and MgCl2
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O, concentration 50 g/L
One plate (three tissues) was used as positive control; each tissue was treated with 30 µL SDS-solution
Test item: Crinipan AD, ground before use; 22.7 mg for each tissue
One plate (three tissues) was used for treatment with the test item.The tissues were wetted with 25 µL DPBS before applying the test item

Experimental performance:
After dosing the tissues with negative control, positive control and test item, they were incubated at 37 °C for 35 min. After 60 min a rinsing step and two incubation periods at 37°C over a total period of 42 h followed.
After incubation, a MTT assay was performed and formazan production was measured by a spectral photometer at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60min

Value:

112.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: percentage values of formazan production in comparison to the negative control

Other effects / acceptance of results:

not applicable
The mean relative absorbance value was increased to 112.7% after the treatment with Crinipan AD. This value is well above the threshold for irritation (50%). Therefore, the test item is considered not irritant. The mean reduction of the relative absorbance of the positive control was 9.0%.

Comparison of formazan production:

For the test item and the positive control the following percentage values of formazan production were calculated in comparison to the negative control:

% Formazan Production

Designation	Crinipan AD	Positive control
% Formazan production (Tissue 1)	93.9%	9.4%
% Formazan production (Tissue 2)	130.5%	7.5%
% Formazan production (Tissue 3)	114.4%	10.3%
% Formazan production Mean	112.7%	9.0%

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance did not show any skin irritation properties in the EpiDerm reconstituted human skin model.

Executive summary:

This in-vitro study human skin model test (EU method B.46) was performed in order to evaluate the potential of crinipan AD to evoke skin irritation in a human-skin-model. The test item crinipan was spread on the surface of three sections of an EpiDerm^TM tissue and incubated for 60 min. The negative control consisting of Dulbecco’s phosphate buffered saline was applied to another three tissue sections. As positive control a SDS-solution (50 g/L) was used for three other tissue sections. After rinsing and two incubation steps of altogether 42 h an enzyme assay to determine cell viability was performed. The tissues were incubated with MTT-reagent for 3 h. The tissues were washed with PBS, dried and transferred to a 24 -well-plate. After incubating with isopropanol at room temperature over night, the developed formazan concentration was measured in a spectral photometer at 570 nm.

The mean percentage values of formazan production were calculated in comparison to the negative control. The mean relative absorbance value was increased to 112.7% after the treatment with crinipan. This value is well above the threshold for irritation (50%). The positive control showed a mean reduction of formazan production of 9.0%.

In conclusion, it can be stated that the test item crinipan is considered as non skin irritant under the experimental conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-06-22 - 2010-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline no. 437 and under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437: "Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants", 2009

Principles of method if other than guideline:

The BCOP (Bovine Corneal Opacity and Permeability) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. The BCOP test uses isolated corneas from freshly slaughtered cattle. Damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an in vitro irritancy score (IVIS), which is used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of a test substance.

GLP compliance:

yes (incl. QA statement)

Species:

other: not applicable (this is an in vitro test)

Strain:

other: not applicable (this is an in vitro test)

Details on test animals or tissues and environmental conditions:

not applicable (this is an in vitro test)

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

mean: 289 mg of crinipan were applied

Duration of treatment / exposure:

4 h at 32 °C

Observation period (in vivo):

not applicable
Opacity of the corneas is measured at 570 nm, using a spectral photometer.
Permeability is determined by the amount of sodium fluorescein dye that penetrates all corneal cell layers. The fluorescein permeability values are measured as optical density at 490 nm using a spectral photometer.

Number of animals or in vitro replicates:

not applicable

Details on study design:

For the BCOP test with crinipan bovine corneas from freshly slaughtered cattle aged 12 to 60 months were used. The eyes of the cattle were transported from the slaughterhouse within 4 h to the test facility in Hank's balanced salt solution supplemented with 0.01% streptomycin and 0.01% penicillin. The corneas were examined and only corneas which were free from defects were used. The dissected corneas were transferred to a cornea holder and incubated with complete minimum essential medium (cMEM) at 32 ± 1°C for 1 h. After the initial incubation, the medium was changed and the baseline opacity was determined.
The test item crinipan was not soluble in any appropriate solvent for the BCOP test and therefore was used neat. For each treatment group (negative control, positive control, test item) three replicates were used. The test item crinipan was applied directly using a weight board. According to the guideline, the "open chamber method" for non-surface-active solids was performed. Averagely, 0.2893 g of the test item was applied on the epithelium in a way that as much as possible of the cornea was covered with test item. The exposition time on the corneas was 10 min at 32 °C. After thorough rinsing with cMEM a post-incubation time of 2 h at 32 °C followed. Then the opacity value of each cornea was determined and the permeability with sodium fluorescein was measured.

Irritation parameter:

in vitro irritation score

Run / experiment:

BCOP max. score: 80

Value:

0

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Calculated IVIS:

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0,9% NaCl	1.7605	1.1190	57.2%
	0.4804
	1.1165
Crinipan AD	0.5106	-0.5610	-165.5%
	-1.1023
	-1.0920
Positive Control 20% Imidazole	74.4592	74.4200	17.5%
	87.4479
	61.3534

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It can be stated that in this study and under the experimental conditions reported, climbazole did not exhibit any ocular irritating potential in vitro.

Executive summary:

The BCOP test was performed to assess the corneal irritation and damage potential of Crinipan AD by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item Crinipan AD was brought onto the cornea of a bovine eye which previously had been incubated with cMEM ( = complete Minimum essential medium) without Phenol red at 32±1°C for one hour and whose opacity had been determined. The test item was incubated on the cornea for four hours at 32±1°C. After removal of the test item, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control, imidazole (20% solution in 0,9% sodium chloride solution) was used as positive control. The positive control induced severe irritation on the cornea, mean IVIS was 74.42. The negative control showed no irritation, mean IVIS was 1.119. The test item was tested neat. A mean IVIS of -0.561 = 0.00 was calculated, corresponding to a classification as not eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The skin irritation properties of crinipan were evaluated in an in vitro study in the human skin model EpiDerm, in a 3-week in vivo skin irritation study in rabbits and a series of 4 skin irritation patch test in human volunteers.

In the in vitro study, which was performed according to EU guideline no. B 46, 3 sections of an EpiDermTM tissue were incubated with crinipan for 60 min. A SDS-solution (50 g/L) and Dulbecco’s phosphate buffered saline were used as positive and negative control, respectively. After rinsing and two incubation steps of altogether 42 h, cell viability was assessed using a MTT enzyme assay. No reduction of cell viability was observed with the test item and the negative control, while the positive control induced a strong decline in cell viability.

Based on these results, crinipan was considered as non-irritating to the skin under the conditions used.

 

In the vivo skin irritation study, crinipan was applied daily to the intact or abraded skin of male and female New Zealand White rabbits for 3 weeks. The test item was applied, either as a 0.5% solution in a hair lotion or as a 0.5% emulsion in polyethylene glycol 400, on 5 d a week for 7 h to the skin of the animals. The amount applied daily was split into two portions applied at an interval of 3.5 h. At the end of each 7-h exposure period, the test item was rinsed off with water. The local effects of the treatment were evaluated using numerical rating scale for skin redness and swelling. Additionally, the test animals were sacrificed at the end of the study and the organs were subjected to gross pathology and histopathology.

Crinipan in either vehicle was relatively well tolerated. However, some degree of skin irritation was observed during the second week of the study with the hair-lotion formulation.

Taken together, the results of these two studies indicate that the skin irritation potential of crinipan is low.

Furthermore, the skin irritating properties of crinipan were evaluated in 4 patch tests in humans.

In general, the study subjects were exposed to the crinipan formulations by semiocclusive application of test patch to the back skin for 24 h or 48 h. SDS of different concentrations (1% to 4%) and demineralized water were used as positive and negative controls, respectively. Treatment sites were visually assessed for irritation at 24 h and at 48 h after patch application.

No or only little effects were observed upon topical application of crinipan and the negative controls in all of these studies, while the positive controls always caused positive reactions.

These results also indicate a low potential of crinipan to cause skin irritation upon topical application. Crinipan was not tested at a maximum concentration of 10%, i.e. at a 20-fold higher concentration than the maximum concentration at which crinipan is used as anti-dandruff agent in cosmetic formulations.

 

Eye irritation:

In the HET-CAM test, crinipan was applied undiluted to the chorioallantoic membrane (CAM) of 9 d-old incubated chicken eggs for 300 s and ocular irritation was assessed via examination of a variety of endpoints (e.g., haemorrhage, lysis, coagulation). SDS (1%) and NaOH (0.1 N) were used as positive controls, NaCl (0.9%) as negative control.

No effects indicative of irritation were observed after application of the test item or the negative control, whereas both positive control substances induced severe irritation.

The BCOP test was performed to assess the corneal irritation and damage potential of crinipan by quantitative measurements of changes in opacity and permeability in a bovine cornea. Corneas of a bovine eyes prerincubated for 1 h and were incubated with the test item for 4 h at 32±1°C. After removal of the test item, opacity and permeability values were measured. Imidazole (20% solution in 0.9% NaCl solution) and physiological NaCl solution were used as positive and negative control, respectively. While the positive control induced severe irritation on the cornea, the test item and the negative control showed no irritation.

The in vivo eye irritation study was conducted with a hair lotion and a polyethylene glycol 400 emulsion, each containing 0.5% of crinipan. A placebo hair lotion and the emulsifier polyethylene glycol 400 were tested in comparison. 100 µL each of the 4 test items was applied into the conjunctival sac of one eye each of 5 and 3 rabbits for 5 min or 24 h, respectively, and were thereafter rinsed off with water. The mucous membranes were then observed for a period of 7 d for redness, swelling and ulceration. After application of the 0.5% solution and hair-lotion placebo the treated eyes showed marked to severe redness and swelling, whereas the 0.5% emulsion of crinipan and the polyethylene glycol 400 were tolerated with virtually no symptoms. On the basis of the test results, it was concluded that the 0.5% crinipan emulsion is no-irritant to eyes of rabbits, however some of the components of the hair-lotion might encompass the irritating effects to eyes observed in the study.

 

Taken together, the results of the two in vitro studies and the in vivo study on the eye irritation properties of crinipan showed that this compound virtually dose not cause any irritation to the mucous membranes of the eyes.

Justification for selection of skin irritation / corrosion endpoint:
The study was a reliable and valid in vitro study acceptable for classification purposes and supported by human patch test data.

Justification for selection of eye irritation endpoint:
The study was a reliable and valid in vitro study acceptable for classification purposes given the lack of any effect

Justification for classification or non-classification

The substance has been tested in vitro for irritative properties in eye irritation tests (the BCOP and the HET-CAM test) and in skin irritation tests (human reconstituted skin model EpiDerm). No effects indicative of any eye or skin irritation potential were observed. The substance did not meet the classification criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC). Therefore, the substance does not have to be classified as skin or eye irritating.

The respiratory irritation potential of crinipan was not studied.