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in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 03 December 2015 (first animal arrival).
Experimental termination date: 04 February 2016 (last day of slide scoring).
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder

Test animals

other: Crl:WI (Han)
Details on test animals or test system and environmental conditions:
Source: Charles River (UK) Ltd., Margate, Kent, CT9 4LT, England
Age: 6 to 7 weeks at start of experiment
Weight: range 160 to 217 g, mean 181 g
Acclimatisation period: at least 5 days
Housing: up to 3 per cage
Diet: pelleted standard diet, ad libitum
Drinking water: tap water, ad libitum
Temperature: 19 to 24 °C
Humidity: 45 to 64% (81% on one occasion only)
Photoperiod: 12 hours darkness to 12 hours light

Administration / exposure

Route of administration:
oral: gavage
0.5% w/v aqueous carboxymethylcellulose with 0.1% v/v Tween 80
Details on exposure:
Vehicle and test substance were administered at a dose volume of 10 mL/kg body weight.
The positive control substance was administered at a dose volume of 5 mL/kg body weight.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Animals received two doses of vehicle alone or test substance in vehicle, approximately 24 hours apart.
Animals treated with the positive control were given a single dose of CPA
Post exposure period:
Animals were observed periodically for 24 hours after the first (all groups) and second dose
(Groups 1 to 4).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Vehicle, negative control
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
CPA, positive control
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):


Tissues and cell types examined:
Slide Analysis: A unique, unambiguous code was devised for each main study animal. Adhesive labels that covered the animal and group identity were affixed to each slide so that the analyst could see only the study number and the new code. 2000 polychromatic erythrocytes (PCE), including micronucleated PCE (MN PCE), were counted for each main study animal. The numbers of normochromatic erythrocytes (NCE) and micronucleated NCE (MN-NCE) were also recorded for the first 1000 cells scored. Only areas of slides of good technical quality and appropriate staining characteristics were scored.
Details of tissue and slide preparation:
Slide Preparation: Range-finder animals were killed after the terminal blood sampling, approximately 24 hours after the second administration of the test item. The main study animals in Groups 1 to 4 were killed approximately 24 hours after the second test item or vehicle administration. Group 5 animals were killed approximately 24 hours after the single administration of the positive Control. A single femur was removed from each animal. The bone marrow cells from the femur were aspirated into labelled tubes and centrifuged. The supernatant was withdrawn and the cells were re suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread. All slides were left to air dry and age overnight before fixing for five minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5% (v/v) Giemsa in Sorensen’s buffer pH 6.0.
Evaluation criteria:
For the test to have been considered positive i.e. a substance was considered to induce clastogenic/aneugenic damage, the following criteria would have had to be met:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded the lab's historical negative Control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result.
Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative, i.e. non-clastogenic/aneugenic, if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared with the negative Controls.
The data were analysed in accordance with the UKEMS guidelines. The data analysed were the proportion of micronucleated PCEs (MN-PCE) and the ratio of polychromatic to normochromatic cells (PCE/NCE).
The proportion of micronucleated PCEs (with respect to the total number of PCEs counted) is considered to be a measure of chromosomal or cell division apparatus damage. The ratio PCE/NCE is considered to provide an estimate of general toxicity of the test item to bone marrow. In this case testing is concerned with a reduction in the PCE/NCE ratio from the vehicle Control value.
The preferred approach for the MN-PCE data was to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative Control. The groups were then compared using a one tailed Fisher Exact test. However, this approach was first validated by carrying out a test for between animal heterogeneity using a chi square test. If the heterogeneity test was significant at the 1% level then an exact Wilcoxon Rank Sum test would be used instead of the Fisher Exact test. The same method was used to compare the positive and negative Controls.
For the PCE/NCE data, the groups given the substance and the positive Control group were compared with the negative Control using one tailed exact Wilcoxon Rank Sum tests.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
Definitive test: There were no clinical signs observed following administration of SYN547407 to male rats at dose levels up to 2000 mg/kg/day, nor were there any adverse clinical observations in Group 1 (negative Control) or Group 5 (positive Control) animals.
Exposure to the substance was confirmed in all range-finder blood samples.

Any other information on results incl. tables

Dose-sighting phase: Rapid breathing was observed following the second administration at 500 mg/kg/day, 1250 mg/kg/day and 2000 mg/kg/day. Pale faeces were also observed following administration at 2000 mg/kg/day. Body weight loss was seen in all animals.
Range-finding phase: Rapid breathing was observed following the second administration at 2000 mg/kg/day. One female showed signs of abnormal sensitivity to touch and disturbance followed by slow breathing and piloerection. Body weight loss was seen in all animals.
Based on the results of this phase, the MTD was considered to exceed the limit dose of 2000 mg/kg/day in males and females.

Applicant's summary and conclusion

The substance is considered to be neither clastogenic nor aneugenic in the in vivo rat bone marrow micronucleus assay.
Executive summary:

The substance was tested under GLP to evaluate its potential to cause damage to chromosomes or cell division apparatus, or to cause cell cycle interference, leading to micronucleus formation in polychromatic erythrocytes in the bone marrow of young adult rats following OECD TG 474.
In all phases, the dosing of the vehicle and test item was by oral (gavage) administration twice, approximately 24 hours apart.
In the dose-sighting phase, three groups of two male rats were given the substance as a suspension in 0.5% w/v aqueous carboxymethylcellulose with 0.1% v/v Tween 80 at 500, 1250 or 2000 mg/kg/day, in order to determine the maximum tolerated dose (MTD). In the range-finding phase, a group of three male and three female rats were given the substance at 2000 mg/kg/day, in order to confirm the MTD. The MTD was confirmed to be greater than the limit dose of 2000 mg/kg/day in male and female rats, and as there was no inter-sex difference in toxicity, the main study was conducted in males only.
A proof of exposure phase was conducted to demonstrate that the bone marrow was exposed to the test item, via analysis of test item in the whole blood of treated animals. The presence of substance was confirmed by analysis of the study samples alongside samples of blank matrix and matrix spiked with the test item. Exposure to the test substance was confirmed in all blood samples.
For the main study phase, three groups, each of six male rats were dosed with 500, 1000 or 2000 mg/kg/day test substance. A group of six male rats (negative controls) was dosed with the vehicle alone and a positive control group, also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA).
Bone marrow was harvested from all range-finding and main study animals approximately 24 hours after the final dose administration and smears were prepared. The stained slides prepared for the main study were coded and 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.
There were no statistically significant increases in micronucleus frequency in male rats treated at any dose level of of the substance, compared with the negative control group. There was no evidence of a statistically significant reduction in the PCE/NCE ratio in male rats treated with the substance and, since proof of exposure to the bone marrow was demonstrated in the range finding phase of the study, this indicated a lack of toxicity of the substance to the bone marrow.
The animals dosed with CPA, the positive control item, had statistically significant increases in the number of micronucleated cells compared with the concurrent vehicle control group, which demonstrated that the test system was capable of detecting a known clastogen and that the scorers were capable of detecting micronuclei. There was a statistically significant decrease in the PCE/NCE ratio in the positive control group, indicating toxicity to the bone marrow.
In conclusion, it can be stated that there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the substance up to the OECD 474 limit dose of 2000 mg/kg/day in male rats. The substance is considered to be neither clastogenic nor aneugenic in the rat bone marrow micronucleus assay.