Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: clastogenicity/aneugenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: minimum 7 weeks
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex per cage, IVC cage (Polysulphone), Type II L
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x per hour
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: 0.9% NaCl
- Justification for choice of solvent/vehicle: The solvent was chosen based on best solubility of the test item and according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 100 mg/mL (1 MTD), 50 mg/mL (0.5 MTD), 20 mg/mL (0.2 MTD)
- Amount of vehicle (if gavage or dermal): 10 mL per kg body weight
- Lot/batch no.: 121672021, 12292410 Braun
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was diluted in 0.9% NaCl within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw.
Frequency of treatment:
The animals received the test item once ip.
Post exposure period:
Sampling of the bone marrow cells was carried out on animals 24 and 48 h after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
three dose groups: 1 MTD (10 mg/kg bw), 0.5 MTD (5 mg/kg bw), 0.2 MTD (2 mg/kg bw)
Basis:
other: solution in 0.9% NaCl
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control: clastogenic control substance, good stability at room temperature, broad basis of historical laboratory data
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
immature erythrocytes of the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. Three animals of each sex were treated ip for detection of the maximum tolerable dose. The maximum dose that was applied was 2000 mg/kg bw according the OECD 474 guideline. The maximum volume which was administered was 10 mL/kg bw. The highest dose group evaluated in the main experiment (100 mg/kg bw) was based on the toxicity observed in the pre-experiment.

METHOD OF ANALYSIS:
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the slides was performed microscopically by using 100 x oil immersion objectives. 2000 immature erythrocytes were scored per animal for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item the ratio between immature and mature erythrocytes was determined. At least 200 erythrocytes were counted per animal and the result was expressed as relative PCE (rel. PCE = proportion of immature (polychromatic) erythrocytes among total erythrocytes).
Evaluation criteria:
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
Statistics:
For the statistics the nonparametric Mann-Whitney Test was used. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
In the pre-experiment a concentration of 200 mg/mL (active component) of the test item was evaluated. One male mouse received a single dose of 2000 mg/kg bw (active component) i.p. and showed toxicity such as reduction of spontaneous activity, constricted abdomen, recumbency, opisthotonos, half eyelid closure and bradykinesia. The animal died within 2 hours after treatment. One male mouse received a single dose of 500 mg/kg bw (active component) i.p. and showed toxicity such as reduction of spontaneous activity, constricted abdomen, opisthotonos, half eyelid closure, recumbency, piloerection, catalepsis, bradykinesia, kyphosis and eye closure. The animal died within 68 hours after treatment. Three male and three female mice received a single dose of 100 mg/kg bw (active component) ip and showed toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, bradykinesia and recumbency.

RESULTS OF DEFINITIVE STUDY
- Toxicity in the main experiment:
100 mg/kg bw was tested as the maximum tolerable dose (1 MTD, 24 h and 48 h) in the main experiment. The volume administered i.p. was 10 mL/kg bw.
All animals treated with the highest dose (1 MTD) showed slight to moderate toxic effects. The animals treated with 50 mg/kg bw (0.5 MTD) showed slight signs of toxicity such as reduction of spontaneous activity, constricted abdomen and half eyelid closure. After 2 hours no toxic symptoms were observed anymore for the animals treated with 0.5 MTD. The animals treated with 20 mg/kg bw (0.2 MTD) showed no signs of toxicity.


- Induction of micronuclei (for Micronucleus assay):
The negative controls (24 h and 48 h) evaluated were within the range of the historical negative control data (0.04 – 0.29%). The mean values of micronuclei observed for the negative control (24 h) were 0.19% (male mice) and 0.13% (female mice). The mean value for the 48 h negative control was 0.10% (male mice) and 0.06% (female mice).
The dose group treated with 0.2 MTD showed mean values of 0.12% (male mice) and 0.09% (female mice). The values observed in both groups were decreased compared to the corresponding negative control, but these decreases were not statistically significant. Additionally, both values were within the range of the historical negative control data. As the observed differences were not statistically relevant decreases with regard to the negative control and observed within the historical negative control data, this effect was not considered biologically relevant.
The dose group treated with 0.5 MTD showed mean values of 0.10% (male mice) and 0.31% (female mice). The value observed in the male group was reduced compared to the corresponding negative control, but this decrease was not statistically significant. Additionally, the value was within the range of the historical negative control data. As the observed difference was a not statistically relevant decrease with regard to the negative control and observed within the historical negative control data, this effect was not considered biologically relevant. The value observed in the female group was increased compared to the corresponding negative control and was slightly over the range of the historical negative control data. However, this increase was not statistically significant. Therefore and as this increase was not confirmed in the higher dose group (1 MTD) this effect was not considered as biologically relevant.
The dose group treated with 1 MTD (24 h treatment) showed mean values of 0.19% (male mice) and 0.12% (female mice). The value observed in the female group was slightly reduced compared to the corresponding negative control, but this decrease was not statistically significant. Additionally, both values were within the range of the historical negative control data and therefore the slight reduction was not considered as biologically relevant.
The mean values observed for the 1 MTD 48 h treatment were 0.11% (male mice) and 0.07% (female mice). The values observed in both groups were slightly increased compared to the corresponding negative control but these increases were not statistically significant. Additionally, both values were within the range of the historical negative control data and therefore the slight increases were not considered as biologically relevant.
No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.


- Ratio of PCE/NCE (for Micronucleus assay):
The negative controls (24 h, 48 h) were within the range of the historical negative control data (0.46 - 0.79). The mean value noted for the 24 h negative control was 0.59 (male mice) and 0.67 (female mice). The mean values detected for the 48 h negative control were 0.69 (male and female mice).
The animal group treated with 0.2 MTD showed mean values for the relative PCE of 0.62 (male mice) and 0.70 (female mice). The mean values observed in the male and in the female group were slightly increased as compared to the corresponding negative control. However, these increases were not statistically significant. Moreover the mean values were within the range of the historical control data and therefore the slight increase was not considered as biologically relevant.
The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 0.71 (male mice) and 0.73 (female mice). The mean values observed in the male and in the female group were slightly increased as compared to the corresponding negative control. However, these increases were not statistically significant. Moreover, the mean values were within the range of the historical control data and therefore the slight increase was not considered as biologically relevant.
The animals who received 1 MTD (24 h treatment) showed mean values of 0.73 (male mice) and 0.71 (female mice). The mean values observed in the male and in the female group were increased as compared to the corresponding negative control. However, these increases were not statistically significant. Moreover the mean values were within the range of the historical control data and therefore the increase was not considered as biologically relevant.
The animal group which was treated with 1 MTD (48 h treatment) showed mean values of the relative PCE of 0.70 (male mice) and 0.73 (female mice). The mean values observed in the male and in the female group were slightly increased as compared to the corresponding negative control. However these increases were not statistically significant. Moreover the mean values were within the range of the historical control data and therefore the slight increases were not considered as biologically relevant.


- Statistical evaluation:
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant differences (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

In a mammalian micronucleus test of murine bone marrow cells, five male and female animals per dose group were treated ip with the test item at doses of 100, 50 and 20 mg/kg bw.  Bone marrow was obtained from the femurs (after animals were sacrificed by cervical dislocation) at 24 h (all dose and control groups) and 48 h (negative control and 1 MTD group) post-treatment.  The vehicle was 0.9% NaCl. The animals received the test item once ip.
There were signs of toxicity during the study.  The animals treated with doses of 0.2 and 0.5 MTD showed no signs of toxicity. The animals treated with 1 MTD showed slight to moderate signs of systemic toxicity such as
reduction of spontaneous activity, bradykinesia, constricted abdomen, recumbency, opisthotonos and half eyelid closure. The substance was tested at an adequate dose based on OECD 474. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.