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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Three Ames assays and two further in vitro and in vivo (in total seven) tests were performed with crinipan. The results indicate the non-genotoxic potential of crinipan under the tests conditions, hence crinipan is considered non-mutagenic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-01-03 to 2007-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline No. 474 and followed the principles of GLP.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2A (1996)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2B (1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SCCNFP Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation (2004)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan (Frederick, MD, USA)
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation:
Toxicity study: males: 25.1-30.0 g
females: 25.6-29.6 g
Supplemetal toxicity study: males: 25.7-30.5 g
females: 23.1-27.6 g
Micronucleus test: males: 34.8-38.3 g

- Assigned to test groups randomly: yes, based on equalization of mean group body weight
- Housing: up to 5 mice of the same sex were housed together in rodent Micro-Barrier cages in an AAALAC-accredited facilty. Heat-treated hardwood chips were used for bedding.
- Diet: certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet), ad libitum
- Water: tap water, ad libitum
- Air change: constantly
- Acclimation period: not less than 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 72±3°F (22.2±1.7°C)
- Humidity: 50±20%
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: polyethylene glycol 200 (PEG 200)
- Justification for choice of vehicle: request of the study sponsor
- Concentration of test material in vehicle: 3.75, 5, 7.5, 01, 15, 20, 30, 40, 50, and 100 mg/mL
- Amount of vehicle: 10 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared freshly just prior to administration as follows:
An appropriate amount of test article for each concentration (3.75, 5, 7.5, 10, 15, 20, 30, 40, 50 and 100 mg/mL) was weighed and combined with an appropriate amount of vehicle and the mixture was vortexed and stirred using a magnetic stir plate/stir bar for ~45 min until a clear, coluorless solution was formed.

Samples of the dose formulations at concentrations of 0 mg/mL (vehicle) and at 3.75, 7.5, and 15 mg/mL were subjected to chemical analysis.
The overall results of these analyses indicate the accuracy of preparation and stability of the formulations used in this study.
Duration of treatment / exposure:
Initial and supplemental toxicity study: Obervation for 3 d after dosing
Micronucleus test: sacrifice at 24 h (all low-dose, medium-dose and positive control animals as well as 5 animals each of the vehicle control and the high-dose group) or 48 h after dosing (the remaining animals of the vehicle control and the high-dose group)
Frequency of treatment:
once (single dose)
Post exposure period:
not applicable
Remarks:
Doses / Concentrations:
Initial toxicity study: 50, 100, 300, 400, 500, and 1000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Supplemental toxicity study: 100, 150, and 200 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Micronucleus test: 37.5, 75, and 150 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Initial and supplemental toxicity study: 3 males and 3 females per group

Micronucleus test: 5 males each at the low and the medium dose level and for the positive control, 10 males for the vehicle control, 15 males at the high dose level
(since no differences in the toxicity between male and female mice were observed in the two dose range finding studies, only males were used in the definitive micronucleus test)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate (CP, CAS number 6055-19-2), obained from Sigma-Aldrich, dissolved in sterile distilled water (5 mg/mL)
- Route of administration: oral, by gavage
- Dose: 50 mg/kg
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose levels for the definite micronucleus test were defined based on the results obtained from the intial and the supplemental toxicity study.

DETAILS OF SLIDE PREPARATION:
At the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labelled centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for 5 min and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were scored using a light microscope and a medium magnification (400X), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the numbers of polychromatic erythrocytes (PCEs) normochromatic erythrocytes (NCEs) and micronuclei were determined.
Two-thousand PCEs per mouse were scored for the presence of micronuclei, resulting in evaluation of a total of 10000 PCEs per each treatment group. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs). The incidence of MNCEs per 2000 PCEs was enumerated for each animal, but the result was not given in the report. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).
Statistics:
The incidence of MNPCEs per 2000 PCEs was determined for each mouse and treatment group.
Statistical significance was determined using one way analysis of variance and Dunnett’s t-test (post hoc) for group mean comparison and regression analysis for detecting any trend using square-root transformed data (square root of MNPCEs/2000 PCEs + 1) using a “p” value of ≤ 0.05.
All analyses were performed separately for each sampling time.
To quantify the test articles effect on erythropoiesis, the proportion of PCEs to total erythrocytes was determined for each animal and treatment group (PCEs/total erythrocyte ratio).
Sex:
male
Genotoxicity:
negative
Remarks:
no significant increase in the frequency of micronucleus formation was observed in any of the treated groups
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF THE INITIAL ANS SUPPLEMENTAL TOXICITY STUDIES
- Dose range: initial toxicity study: 50-1000 mg/kg; supplemental toxicity study: 100-200 mg/kg
- Solubility: The test article formed a clear, colourless solution in the vehicle at concentrations of up to 100 mg/mL, the maximum concentration tested in this study.
- Clinical signs of toxicity in test animals:
Mortality was observed in 1/3 males at 150 mg/kg, 1/3 males at 200 mg/kg, 2/3 females at 300
mg/kg, 1/3 males and 1/3 females at 400 mg/kg, 1/3 males and 2/3 females at 500 mg/kg, and 3/3
males and 1/3 females at 1000 mg/kg. Clinical signs of toxicity (e.g., hyperactivity, prostration, irregular breathing, and excessive salivation) were observed at all concentrations of the test article, increasing in severity with dose. Reductions in mean (group) body weights, up to 19.5%, were observed in some of the test article treated groups.
- Rationale for exposure: Determination of appropriate dose levles for the definite micronucleus test.


RESULTS OF DEFINITIVE MICRONUCLEUS TEST
- Induction of micronuclei: No significant increase in the incidence of MNPCEs was observed in any of the test article-treated groups relative to the respective vehicle control groups at 24 or 48 h after dose administration.
- Ratio of PCE/NCE: No appreciable reductions in the ratio of PCE to NCE were observed in the test article-treated groups relative to the respective vehicle control groups.
Signs of toxicity:
One male mouse (1/15) at 150 mg/kg was found dead at 24 h post dose. In addition to mortality, clinical signs of toxicity were observed in all test article treated groups, increasing in severity with dose (Tables 4 and 5). Reductions in mean (group) body weights up to ~ 10%, were observed in some of the test article treated groups.

Bone marrow micronucleus analysis

 Treatment  Time (h)  Number of mice   PCE/total erythrocytes (mean)     PCE/total erythrocytes SD   Micronucleated PCE per 1000 (mean)     Micronucleated PCE per 1000 SD  Significance
 PEG 200  24  5 M  0.542  0.05  0.5  0.5  -
 37.5 mg/kg Climbazole  24  5 M  0.570  0.05  0.2  0.27  NS
 75 mg/kg Climbazole  24  5 M  0.497  0.05  0.2  0.27  NS
 150 mg/kg Climbazole 24  5 M  0.507  0.06  0.4  0.42 NS 
 50 mg/kg Cyclophosphamide  24  5 M  0.590  0.02  17.0  3.55  p<=0.05
 PEG 200  48  5 M  0.443  0.08  0.1  0.22  -
 150 mg/kg Climbazole  48  5 M  0.421  0.05  0.4  0.22  NS
Conclusions:
Interpretation of results (migrated information): negative
In an in vivo micronucleus test in male ICR mice, animals received a single oral dose of the test item in polyethylene glycol 200 at dose levels of 37.5, 75, and 150 mg/kg. Upon bone marrow analysis after 24 and 48 hours, no significant increase in the incidence of polynucleated MNPCEs was observed and the test substance was judged to be negative in this assay.
Executive summary:

The genotoxic potential of crinipan (climbazole) was evaluated in an in vivo micronucleus test in male ICR mice. After a dose of 150 mg/kg habe been determined as the maximum tolerated dose in two preliminary toxicity studies in male and female ICR mice, groups of male ICR mice received a single oral administration of the test item in polyethylene glycol 200 at dose levels of 37.5, 75, and 150 mg/kg. Cyclophosphamide monohydrate (50 mg/kg) was used as positive control. At 24 or 48 h after adminstration, the animals were sacrificed, slides were prepared from their bone marrow cells, and the slides were scored for the occurence of micronuclei. The test item did not induce any significant increase in the frequency of micronucleus formation. Thus, crinipan (climbazole) was found to be negative under the conditions used in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Three Ames assays and each two further in vitro and in vivo (in total seven) tests were performed with crinipan. The results indicate the non-genotoxic potential of crinipan under the tests conditions, hence crinipan is considered non-mutagenic.

 

Three Ames tests were performed using crinipan:

1) Ames test (key study) - according to OECD 471 and current GLP regulation. Crinipan (up to 5000 µg/plate) was tested using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of metabolic activation by rat liver S9 mix. The assay was performed using both the plate incorporation and the preincubation method. Under the conditions of this study, crinipan was concluded to be negative in all tester strains in the presence and absence of metabolic activation.

2) Ames test (supportive study) - no test guideline or GLP regulation followed. The potential mutagenicity of crinipan (up to 12500 µg/plate) was investigated in the S. typhimurium tester strains TA98, TA100, TA1535, and TA1537, both in the presence and in the absence of metabolic activation by rat S9 mix. The test item was found to be cytotoxic to the tester strains ≥1000 µg/plate, and precipitation of the test item was observed ≥2000 µg/plate. Crinipan was not mutagenic in any of the tester strains.

3) Ames test (supportive study) - no test guideline or GLP regulation followed. The potential mutagenicity of crinipan (up to 2000 µg/plate) was evaluated in the S. typhimurium tester strains TA98, TA100, and TA1537, both in the presence and in the absence of metabolic activation by rat liver S9 mix. No cytotoxicity was observed with metabolic activation, slight cytotoxicity was observed in the tester strains TA100 and TA1537 at the highest concentration in the presence of metabolic activation. Precipitation of the test compound was observed ≥315 µg/plate. Crinipan was not mutagenic in any of the tester strains.

These three assays confirmed non mutagenicity of crinipan under the test conditions.

 

Apart from three Ames assays two other in vitro and two in vivo tests were also performed with crinipan.

 

In vitro micronucleus assay in human peripheral blood lymphocytes:

The genotoxic potential of the test item, crinipan USP (climbazole) was evaluated in an in vitro micronucleus assay in human peripheral blood lymphocytes, both in the presence and in the absence of metabolic activation by rat S9 mix. The assay was performed according to OECD draft guideline No. 487. In three different experiments, cells were exposed to concentrations of the test item ranging from 160 to 210 µg/mL and 20 to 85 µg/mL for tests with and without metabolic activation, respectively. Additionally, two different period of stimulation with PHA (24 and 48 h) prior to the treatment of the cells with the test item were used. 4-nitroquinoline-N-oxide (2.5 µg/mL), vinblastine (0.06 or 0.08 µg/mL), and cyclophosphamide (6.25 µg/mL) were used as positive controls. In most cases, treatment of the cells with the test item did not induce significant increase in the number of micronucleated cells, as compared to vehicle controls. However, small but statistically significant increases in the frequency of micronucleated cells over the levels observed in the respective vehicle controls were obtained in experiment 2 (with metabolic activation, with 48 h PHA stimulation) and in experiment 3 (without metabolic activation, with 24 h of PHA stimulation). However, as these increases were observed only at the highest concentrations of the test item (200 and 85 µg/mL, respectively), where relativly high levels of cytotoxicity occurred (reduction in in RI of 63 and 59%, respectively) and since the increases were not reproduced in the other experiments and did exceed the significance limit only by a narrow margin, these results were considered not biologically relevant. Hence, the overall outcome of the study was judged to be negative. Thus, the results of this study indicate that the test item is not genotoxic to human peripheral blood lymphocytes under the conditions employed in this assay.

 

In vitro mouse lymphoma assay in L5178Y/TK+/-cells:

In this assay, which was conducted according to OECD 476 and GLP guidelines, test cells were exposed to concentrations of crinipan ranging from 5 - 80 µg/mL for 4 and 24 h both in the presence and in the absence of metabolic activation by rat S9 mix. 2,7-Dimethylbenzanthracene and methylmethanesulfonate were used as positive controls for experiments with and without metabolic activation, respectively. When using the 4-h exposure period, the test item did not induce any significant increase in the frequency of mutations in the TK gene, both in the presence and in the absence of metabolic activation. However, when the 24-h exposure was used, exposure of the test cells in the absence of metabolic activation led to a significant increase in mutation frequencies. Accordingly, the results of this study indicate that the test item was not mutagenic to the tester cells when applied for 4 h, but it showed some mutagenic activity when the cells were exposed to it for the longer time period of 24 h.

 

In vivo unscheduled DNA synthesis (UDS) test in Sprague-Dawley rats:

After appropriate dose levels had been determined in a preliminary dose-range finding study in male and female rats, the definite UDS test was performed according to OECD 486 and GLP guidelines by administering crinipan to groups of 10 males per dose at single oral doses of 100 and 200 mg/kg. Dimethylnitrosamine (35 mg/kg) was used as positive control. 3 animals of each group were sacrificed at 2 to 4 h and at 12 to 16 h after dosing, and hepatocytes were isolated from the livers of the animals. The hepatocytes were maintained in cell culture medium, exposed to [3H]-thymidine (10 µCi/mL) for 17 to 20 h. After work-up, the cells were fixed, mounted up on glass slides and the slides were analyzed for incorporation of [3H]-thymidine into nucleic DNA using autoradiography. Crinipan did not induce any significant increase in the incorporation of radioactive thymidine into hepatocytes isolated from treated rats, and it was thus concluded that crinipan was negative in vivo UDS test when tested up to the maximum dose level of 800 mg/kg.

 

In vivo micronucleus test in male ICR mice:

The study was performed according to OECD 486 and current GLP guidelines. Dose of 150 mg/kg crinipan have been determined as the maximum tolerated dose in two preliminary toxicity studies in male and female ICR mice. Groups of male ICR mice received a single oral administration of crinipan in polyethylene glycol 200 at dose levels of 37.5, 75, and 150 mg/kg. Cyclophosphamide monohydrate (50 mg/kg) was used as positive control. At 24 or 48 h after administration, the animals were sacrificed, slides were prepared from their bone marrow cells, and the slides were scored for the occurrence of micronuclei. The test item did not induce any significant increase in the frequency of micronucleus formation. Thus, crinipan was found to be negative under the conditions used in this assay.

 

From these in vitro and in vivo assays, it is confirmed that crinipan does not exhibit genotoxic potential.


Justification for selection of genetic toxicity endpoint
GLP and guideline study performed in mammalian cells to detect the activity of clastogenic and aneugenic chemicals

Justification for classification or non-classification

The substance does not have to be classified for mutagenicity according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) for the following reasons:

- it did not reveal any mutagenic effect in three bacterial reverse mutation assays in the presence or absence of metabolic activation,

- did not cause biologically relevant increases in an in vitro micronucleus test in human peripheral lymphocytes,

- caused mutagenic effects in the in vitro mouse lymphoma assay in L5178Y TK+/- cells, but

- did not induce increased numbers of micronuclei in an in vivo bone marrow micronucleus test in male ICR mice, and

- did not cause unscheduled DNA synthesis in an in vivo UDS test in male and female Sprague-Dawley rats.