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:
2013-03-26 to 2013-04-17
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:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
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
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name: Carbamic acid, N-(3-(triethoxysilyl)propyl)2-(ethyl-(4-nitrosophenyl) amino) ethyl ester
- Internal code: SAT 120009
- Batch No.: 12/34#
- CAS: 1195231-94-7
- Purity: 92.8 wt% (NMR), 93.3 area% (HPLC)

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
- Fasting period before study:
- 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(s)/solvent(s) used: Cottonseed Oil.
- 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: 10 mg/mL (1 MTD), 5 mg/mL (0.5 MTD), 2 mg/mL (0.2 MTD)
- Amount of vehicle (if gavage or dermal): 10 mL per kg body weight
- Lot/batch no. (if required): MKBJ0602V Sigma
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was grinded thoroughly in a mortar and suspended in Cottonseed Oil within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw.
Duration of treatment / exposure:
44 h and 68 h
Frequency of treatment:
The animals received the test item once ip.
Post exposure period:
Sampling of the peripheral blood was carried out on animals 44 h (all control and dose groups) and 68 h (negative control, 1 MTD group) after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
three dose groups: 1 MTD (100 mg/kg bw), 0.5 MTD (50 mg/kg bw), 0.2 MTD (20 mg/kg bw)
Basis:
other: suspension in Cottonseed Oil
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:
peripheral blood erythrocytes
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:
Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity were recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI respectively. At least 10000 immature erythrocytes per animal were scored 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. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) 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
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

RESULTS OF RANGE-FINDING STUDY

Clinical signs of toxicity in test animals:

In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. One male mouse received a single dose of 2000 mg/kg bw ip and showed strong toxicity such as reduction of spontaneous activity, recumbency, constricted abdomen, opisthotonos, ataxia, bradykinesia, piloerection, tachypnoea and half eyelid closure. The animal died 3 hours after treatment. One male mouse received a single dose of 1000 mg/kg bw ip and showed strong toxicity such as reduction of spontaneous activity, recumbency, constricted abdomen, opisthotonos, bradykinesia, muscle twitches, tachypnoea and half eyelid closure. The animal died 2 hours after treatment. One male mouse received a single dose of 200 mg/kg bw ip and showed strong toxicity such as reduction of spontaneous activity, catalepsis, constricted abdomen, opisthotonos, bradykinesia, piloerection and half eyelid closure. Based on animal welfare aspects the animal was euthanized 20 hours after the treatment. Three male and three female mice received a single dose of 100 mg/kg bw ip and showed toxicity such as reduction of spontaneous activity, recumbency, constricted abdomen, piloerection, opisthotonos, ataxia, bradykinesia, kyphosis, catalepsis, eye closure and half eyelid closure.

RESULTS OF DEFINITIVE STUDY

Toxicity in the main experiment:

100 mg/kg bw was tested as the maximum tolerable dose (1 MTD) in the main experiment. The volume administered was 10 mL/kg bw ip.

All animals treated with the highest dose group (1 MTD) showed moderate toxic effects such as reduction of spontaneous activity, prone position, constricted abdomen, piloerection, half eyelid closure, eye closure, opisthotonos and catalepsis. Animals treated with 50 mg/kg bw (0.5 MTD) showed the same signs of toxicity as mentioned for the 1 MTD dose group animals, except prone position. These signs of toxicity were in total less intensely developed (mild/moderate) than in the 1 MTD group animals. After 24 h no toxic symptoms were observed anymore. The animals treated with 20 mg/kg bw (0.2 MTD) showed within the first hour the same toxic effects as the 1 MTD dose group, except prone position and eye closure. These signs of toxicity were mildly expressed. After two hours only a mild reduction in the spontaneous activity and a mild piloerection remained. After 24 h no toxic symptoms were observed anymore.

Induction of micronuclei (for Micronucleus assay):

The negative controls (44 h and 68 h) evaluated were within the range of the historical control data of the negative control (0.08 – 0.34%). The mean values of micronuclei observed for the negative control (44 h) were 0.26% (male mice) and 0.17% (female mice). The mean values for the 68 h negative control were 0.23% (male mice) and 0.16% (female mice).

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.22% (male mice) and 0.19% (female mice). The values were within the range of the corresponding negative control as well as within the range of the historical negative control data.

The mean values noted for the 0.5 MTD dose group were 0.18% (male mice) and 0.24% (female mice). The value observed in the male group was slightly decreased as compared to the corresponding negative control. However, this decrease was not statistically significant. The values observed in the female group were within the range of the corresponding negative control data. Both values were within the range of the historical negative control data.

The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.18% (male mice) and 0.27% (female mice). The value observed in the male group was statistically significantly decreased compared to the corresponding negative control. The value observed in the female group was slightly increased compared to the corresponding negative control data. However both values were within the range of the historical negative control data.

The mean values observed for the 1 MTD 68 h treatment were 0.16% (male mice) and 0.19% (female mice). The value observed in the male group was statistically significantly decreased compared to the corresponding negative control. However, the value was within the range of the historical negative control data. The value observed in the female group was within the range of the corresponding negative control data as well as within the range of the historical negative control data.

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 (44 h, 68 h) were within the range of the historical control data of the negative control (1.19 - 4.30). The mean values noted for the 44 h negative control were 2.03 (male mice) and 1.15 (female mice). The mean values detected for the 68 h negative control were 2.03 (male mice) and 1.70 (female mice).

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.69 (male mice) and 1.67 (female mice). The value observed in the male group was slightly decreased and the value noted in the female group was slightly increased as compared to the corresponding negative control. However this decrease/increase was not statistically significant. Moreover the mean values were within the range of the historical control data.

The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 1.69 (male mice) and 1.51 (female mice). The value observed in the male group was slightly decreased and the value noted in the female group was slightly increased as compared to the corresponding negative control. However this decrease/increase was not statistically significant. Moreover the mean values were within the range of the historical control data.

The animals who received 1 MTD (44 h treatment) showed mean values of 1.46 (male mice) and 1.42 (female mice). The value observed in the male group was slightly decreased and the value noted in the female group was slightly increased as compared to the corresponding negative control. However this decrease/increase was not statistically significant. Moreover the mean values were within the range of the historical control data.

The animal group which was treated with 1 MTD (68 h treatment) showed mean values of the relative PCE of 1.24 (male mice) and 1.63 (female mice). The value observed in the male group was slightly decreased and the value noted in the female group was slightly increased as compared to the corresponding negative control. However this decrease/increase was not statistically significant. Moreover the mean values were within the range of the historical control data.

Statistical evaluation:

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated, except the values observed for the 1 MTD male groups (44 h and 68 h). These groups were significantly decreased as compared to the corresponding negative controls. However the values were within the range of the historical negative control data. Based on this data these decreases were regarded as not biologically relevant.

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 SAT 120009 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, SAT 120009 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

SUMMARY

In a NMRI mouse peripheral blood micronucleus assay, five male and female animals per dose group were treated ip with SAT 120009 at doses of 20, 50 and 100 mg/kg bw.  Peripheral blood cells were harvested at 44 h (all dose and control groups) and 68 h (negative control and 1 MTD group) post-treatment.  The vehicle was Cottonseed Oil. 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 dose-depended mild to moderate signs of systemic toxicity. The animals treated with 1 MTD showed moderate signs of systemic toxicity such as reduction of spontaneous activity, prone position, constricted abdomen, piloerection, half eyelid closure, eye closure, opisthotonos and catalepsis. SAT 120009 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.