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:
2012-03-27 to 2012-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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(s)/solvent(s) used: physiol. saline
- 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: 50 mg/mL (1 MTD), 25 mg/mL (0.5 MTD), 10 mg/mL (0.2 MTD)
- Amount of vehicle: 10 mL per kg body weight
- Lot/batch no. (if required): 111209, 111036 AlleMan Pharma
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was prepared and suspended 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 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 (500 mg/kg bw), 0.5 MTD (250 mg/kg bw), 0.2 MTD (100 mg/kg bw)
Basis:
other: suspension 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 control 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 (500 mg/kg bw) 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 valid:
yes
Positive controls valid:
yes
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 of the test item was evaluated. One male mouse received a single dose of 2000 mg/kg bw ip and showed toxicity, such as reduction of spontaneous activity, half eyelid closure, opisthotonos, kyphosis, bradykinesia, piloerection and abnormal breathing. The animal died 4 hours after the treatment. Three male and three female mice received a single dose of 500 mg/kg bw ip and showed toxicity, such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, bradykinesia, kyphosis, opisthotonos, recumbency and muscle twitches. Additionally, the fur and urine of the animals were red coloured by the test item.


RESULTS OF DEFINITIVE STUDY
- 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 – 0.43%). The mean values of micronuclei observed for the negative control (44 h) were 0.28% for male and 0.25% for female mice. The mean values for the 68 h negative control were 0.24% for the male and 0.29% for the female mice.
The mean values of micronuclei observed after treatment with 0.2 MTD were 0.23% for male and 0.22% for female mice. These values were within the range of the corresponding negative control and within the range of the historical negative control data.
The mean values noted for the 0.5 MTD dose group were 0.22% for male and 0.23% for female mice. These values were within the range of the corresponding negative control and within the range of the historical negative control data.
The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.20% for male and 0.20% for female mice. These values were within the range of the corresponding negative control. Both values were within the range of the historical negative control data.
The mean values observed for the 1 MTD 68 h treatment were 0.26% for the male and 0.17 for the female mice. The value observed in the male group was within the range of the corresponding negative control. The value observed in the female group was significantly decreased compared to the corresponding negative control. Both values were 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 negative control data (1.19-3.97), except for the value of the negative control of male mice (68 h) which was slightly above the range of the historical control data. This slight increase can be attributed to the blood loss of the animals 24 h earlier. The mean values noted for the 44 h negative control were 3.88 for the male and 2.60 for the female mice. The mean values detected for the 68 h negative control were 3.99 for the male and 3.39 for the female mice.
The animal group treated with 0.2 MTD showed mean values of the relative PCE of 3.70 (male mice) and 1.80 (female mice). The mean values observed in the male and in the female group were decreased compared to the corresponding negative control, but the decrease was not statistically significant. Moreover, the 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 3.17 (male mice) and 2.71 (female mice). The mean value observed in the male group was decreased and the value observed in the female group was increased compared to the corresponding negative control, but these changes were not statistically significant. Moreover, the values were within the range of the historical control data.
The animals who received 1 MTD (44 h treatment) showed mean values of 3.41 (male mice) and 3.51 (female mice). The mean value observed in the male group was decreased and the value observed in the female group was increased compared to the corresponding negative control, but these changes were not statistically significant. Moreover, the 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 4.09 (male mice) and 3.55 (female mice). The values observed in the male and female group were increased compared to the corresponding negative control, but these changes were not statistically significant

- 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.

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 FAT 40858/A TE did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item FAT 40858/A TE is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

In a NMRI mouse peripheral blood micronucleus assay, five male and female animals per dose group were treated ip with FAT 40858/A TE at doses of 100, 250 and 500 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 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 slight and moderate signs of systemic toxicity, respectively. The animals treated with a dose of 1 MTD showed signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, half eyelid closure, eye closure, bradykinesia, catalepsis, recumbency and opisthotonos. FAT 40858/ A TE 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 asacceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.