[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

in vitro chromosome aberration test (OECD TG 473): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 31 August 2016 and 22 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Experiment 1:
The following dose levels were used: in all strains both with and without S9 - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (the maximum recommended dose level)

- Experiment 2:
Based on the results of Experiment 1 the dose range used for all strains in Experiment 2 was 15 to 5000 µg/plate, both with and without S9 activation.

Vehicle / solvent:

- Solvent used: Acetone
The test item was immiscible in both sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Acetone was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

(untreated plates)

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: direct plate incorporation method
- Experiment 2: change in test methodology from plate incorporation to pre-incubation.


DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain

DETERMINATION OF CYTOTOXICITY
All of the plates were incubated at 37 ± 3”C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

For the test substance to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose-levels or at the highest practicable dose-level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test item precipitate (globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:

Interpretation of results:
FRET 13-0460 was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 g/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations.  The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate.

Six test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.  There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test.  Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).  A test item precipitate (globular in appearance) was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).  Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).

Conclusion

FRET 13-0460 was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 01 September 2016 and 29 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch No. RB390-01
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 16 June 2016
- Purity: 99.4%
- Storage condition of test material: Approximately 4”C in the dark
- Appearance: Clear colorless liquid

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose range of test item used was 0, 8.20, 16.41, 32.81, 65.63, 131.25, 262.5, 525, 1050 and 2100 µg/mL

Main study:
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 22.5, 30, 60 and 180 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 15, 30, 45, 60, 90, 120 and 135 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 45, 60 and 75 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 30 years
Main Experiment: female, aged 25 years
Main Experiment Repeat: female, aged 24 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No’s PB/NF S9 10/04/16 (preliminary toxicity test only) and PB/NF 25/08/16 were used in this study. A copy of the S9 Certificates of Efficacy are presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The molecular weight of the test item was given as 210, therefore, the maximum dose level was 2100 µg/mL, which was calculated to be equivalent to 10 mM, the maximum recommended dose level. However, the maximum concentration should have designated at 2000 µg/mL not 2100 µg/mL because the molecular weight was greater than 200. Therefore, the maximum dose level in culture was slightly above that recommended by the OECD 473 Test Guideline of 2000 µg/mL (the 2 mM equivalent dose level) in the preliminary toxicity test only.
The purity of the test item was 99.4% and was not accounted for in the test item formulations.
The test item was insoluble in MEM at 21 mg/mL and DMSO at 105 and 210 mg/mL. However, the test item was miscible in acetone at 210 and 420 mg/mL in solubility checks performed in house.
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µl aliquots. Consequently, the maximum concentration in solution was 2100 µg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991)

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 8.20, 16.41, 32.81, 65.63, 131.25, 262.5, 525, 1050 and 2100 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 22.5, 30, 60 and 180 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 15, 30, 45, 60, 90, 120 and 135 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 45, 60 and 75 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
Coding
The slides were coded using a computerized random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors

Evaluation criteria:

Data Evaluation
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed. However, for CP 2 µg/mL ‘A’ culture, 150 cells were assessed from one slide and 54 cells were assessed from the parallel slide prepared from the same culture. Consequently, a total of 204 cells were analyzed due to a poor positive response in the first 150 cells. Both slides were coded and scored by different technicians and the results from both slides were combined so the ‘A’ culture of CP µg/mL could meet the acceptability criteria of the OECD 473 test guideline.

Statistics:

Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 8.20 to 2100 µg/mL. The molecular weight of the test item was given as 210, therefore, the maximum dose level was 2100 µg/mL, which was calculated to be equivalent to10 mM, the maximum recommended dose level. However, the maximum concentration should have designated at 2000 µg/mL not 2100 µg/mL because the molecular weight was greater than 200. Therefore, the maximum dose level in culture was slightly above that recommended by the OECD 473 Test Guideline of 2000 µg/mL (the 2 mM equivalent dose level) in the preliminary toxicity test only.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 65.63 µg/mL in exposure groups in the absence of metabolic activation (S9) and at and above 131.25 µg/mL in the exposure group in the presence of S9.
Hemolysis was observed following exposure to the test item at and above 65.63µg/mL in the 4(20)-hour exposure group in the absence of S9 only and at and above 131.25 in the 24-hour continuous exposure group. In the (20)-hour exposure group in the presence of S9, hemolysis was observed at 65.63 µg/mL to 525 µg/mL only. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 32.81 µg/mL in the 4(20)-hour exposures in the presence and absence of S9. The maximum dose with metaphases present in the 24 hour continuous exposure was 65.63 µg/mL. The mitotic index data are presented in Table 1. The test item induced marked evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was limited on toxicity in all three exposure groups.

Chromosome Aberration Test – Main Experiment
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of FRET 13-0460 (µg/mL)
4(20)-hour without S9 0*, 7.5, 15*, 22.5*, 30*, 60, 180, MMC 2*
4(20)-hour with S9 (2%) 0*, 15, 30*, 45*, 60*, 90, 120, 135, CP 2*
24-hour without S9 0*, 7.5, 15, 30*, 45*, 60*, 75, MMC 0.1*

* = Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level in the 4(20)-hour exposure group in the presence of S9 and the 24-hour continuous exposure group. The maximum dose level of test item with metaphases suitable for scoring in the 4(20)-hour exposure group in the absence of S9 was 30 µg/mL.
No precipitate observations were made at the end of exposure in any of the exposure groups. Haemolysis was observed at and above 22.5 µg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 45 µg/mL in the 4(20)-hour exposure group in the presence of S9 and at 75 µg/mL in the 24-hour continuous exposure group. In addition to the hemolysis observations, a reduced cell pellet was observed at and above 60 µg/mL in the 4(20)-hour exposure groups only.
The mitotic index data for the Main Experiment are given in Table 2 and Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups.
With the absence of S9, in the 4(20)-hour exposure group, 19% and 46% mitotic inhibition was achieved at 22.5 and 30 µg/mL, respectively. Above this dose level, there were no scorable metaphases present for assessment. Therefore, the maximum dose level selected for metaphases analysis was 30 µg/mL because it approached optimum toxicity. In the 24-hour continuous exposure group, 46% mitotic inhibition was observed at 60 µg/mL. Few metaphases were present at 75 µg/mL, thetherefore, 60 µg/mL was selected as the maximum dose level for metaphase analysis.
In the presence of S9, an inhibition of mitotic index of 23% and 51% was noted at 45 and 60 µg/mL, respectively. Above this dose level, there were very few metaphases available for assessment. Consequently, 60 µg/mL was selected as the maximum concentration for metaphase analysis.

The assay was considered valid as it met all of the following criteria:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
• All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed. However, for CP 2 µg/mL ‘A’ culture, 150 cells were assessed from one slide and 54 cells were assessed from the parallel slide prepared from the same culture. Consequently, a total of 204 cells were analyzed due to a poor positive response in the first 150 cells. Both slides were coded and scored by different technicians and the results from both slides were combined so the ‘A’ culture of CP µg/mL could meet the acceptability criteria of the OECD 473 test guideline.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a statistically significant increase in polyploid cells at any dose level in any of the exposure groups.

Tale 1 Mitotic Index - Preliminary Toxicity Test

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	3.85	100	1.90	100	4.60	100
8.20	4.25	110	2.80	147	2.95	64
16.41	2.30	60	3.95	208	3.10	67
32.81	0.70	18	3.15	166	4.10	89
65.63	NM P H	-	NM H	-	2.45 P	53
131.25	NM P H	-	NM P H	-	NM P H	-
262.5	NM P H	-	NM P H	-	NM P H	-
525	NM P H	-	NM P H	-	NM P H	-
1050	NM P H	-	NM P	-	NM P H	-
2100	NM P H	-	NM P	-	NM P H

-         = Not assessed for mitotic index

NM    = No or too few metaphases or insufficient metaphases suitable for scoring

P        = Precipitate observed at end of exposure period in blood-free cultures

H       = Hemolysis observed at the end of exposure in blood cultures

Table 2 Mitotic Index - Main Experiment (4(20)-hour Exposure Groups)

Dose Level (µg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	4.15	4.80	4.48	100	5.80	4.85	5.33	100
7.5	3.30	4.45	3.8	87	NA	NA	NA	NA
15	4.10	5.50	4.80	107	5.35	6.45	5.90	111
22.5	3.40 H	3.85 H	6.63	81	NA	NA	NA	NA
30	2.45 H	2.40 H	2.43	54	5.35	5.95	5.65	106
45	NA	NA	NA	NA	3.65 H	4.55 H	4.10	77
60	NM H R	NM H R	-	-	2.00 H	3.20 H	2.60	49
90	NA	NA	NA	NA	NM H R	NM H R	-	-
120	NA	NA	NA	NA	NM H R	NM H R	-	-
135	NA	NA	NA	NA	NM H R	NM H R	-	-
180	NM H R	NM H R	-	-	NA	NA	NA	NA
NMC 0.2	2.65	2.55	2.60	58	NA	NA	NA	NA
CP 2	NA	NA	NA	NA	3.90	3.70	3.80	71

MMC  = Mitomycin C

CP       = Cyclophosphamide

NA      = Not applicable

-        = Not assessed for mitotic index

NM     = No or too few metaphases suitable for scoring

H        = Hemolysis

R        = Reduced cell pellet

Table 3 Mitotic Index - Main Experiment (24 -hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	4.40	4.35	4.38	100
7.5	-	-	-	-
15	5.40	7.20	6.30	144
30	6.05	4.75	5.40	123
45	3.25	5.15	4.20	96
60	1.85	2.85	2.35	54
75	NM H	NM H	-	-
MMC 0.1	2.55	2.10	2.33	53

MMC      = Mitomycin C

-              = Not assessed for mitotic index

NM         = Too few metaphases suitable for scoring

H             = Hemolysis

Conclusions:

FRET 13-0460 did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells.  It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991).  

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls.  In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited primarily on toxicity.  The dose levels selected for the Main Test were as follows:

Group	Final concentration of test item FRET 13-0460 (µg/mL)
4(20)-hour without S9	0, 7.5, 15, 22.5, 30, 60, 180
4(20)-hour with S9 (2%)	0, 15, 30, 45, 60, 90, 120, 135
24-hour without S9	0, 7.5, 15, 30, 45, 60, 75

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that either induced or approached 55±5% mitotic inhibition.

Conclusion

The test item, FRET 13-0460 was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test substance was found to be non-mutagenic in a bacterial reverse mutation assay (Ames test) and and non-clastogenic in vitro chromosome aberration test.