Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES

FRET 13-0545 was considered to be non-mutagenic under the conditions of this test following OECD 471 "Bacterial Reverse Mutation Test".

In vitro chromosome aberration

The test item, FRET 13-0545, was, considered to be non-clastogenic to human lymphocytesin vitro according to the OECD 473 Guideline the Mammalian Chromosome Aberration Test method (human peripheral blood lymphocytes).

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 10 October 2016 and 24 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, in compliance with GLP, without deviations that influence the quality of the results.

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
Escherichia coli (WP2uvrA): tryptophan locus

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

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:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
TA100 (without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
TA1535 (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
WP2uvrA (with and without S9-mix), TA98 and TA1537 (with S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate.
TA100 (with S9-mix), TA98 and TA1537 (without S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
Up to eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to
pre-incubation.

Vehicle / solvent:

- Solvent used: DMSO
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

(untreated plates)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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:

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

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.

In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 µg/plate in the absence of S9-mix and to Salmonella tester strains TA100 and TA1535 initially from 1500 µg/plate in the presence S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA (absence or presence of S9-mix), TA1537 and TA98 (presence of S9-mix). Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix. In the second mutation test (pre-incubation method), the test item induced a stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA100), 500 µg/plate (TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100 and TA98). No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of
S9-mix.

There were no toxicologically 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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 5000 µg/plate in the first mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A single count for WP2uvrA (vehicle control dosed in the absence of S9-mix after the second mutation test) was just below the minimum historical untreated/vehicle control profile. This count was still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9 mix. 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:

FRET 13-0545 was considered to be non-mutagenic under the conditions of this test in AMES following OECD TG 471.

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 µg/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 ranged between 0.5 to 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Up to eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally 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.

In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 500 µg/plate in the absence of S9-mix and to Salmonella tester strains TA100 and TA1535 initially from 1500 µg/plate in the presence S9-mix.  No toxicity was noted to Escherichia coli strain WP2uvrA (absence or presence of S9-mix), TA1537 and TA98 (presence of S9-mix).  Consequently, the toxic limit or the maximum recommended dose level (5000 µg/plate) of the test item was employed in the second mutation test, depending on bacterial tester strain and absence or presence of S9-mix.  In the second mutation test (pre-incubation method), the test item induced a stronger toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate (TA100), 500 µg/plate (TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA).  In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA100 and TA98).  No toxicity was noted to Escherichia coli strain WP2uvrA dosed in the presence of S9-mix.  The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no toxicologically 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).  A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 5000 µg/plate in the first mutation test.  This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted.  Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Conclusion

FRET 13-0545 was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 October 2016 and 19 January 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 473 using the Mammalian Chromosome Aberration Test and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mammalian Chromosome Aberration Test

Specific details on test material used for the study:

Identification: FRET 13-0545
Physical state/Appearance: Clear colorless liquid
Batch: PDJ 378-68
Purity: 93.2%
Expiry Date: 30 June 2018
Storage Conditions: Approximately 4 °C in the dark
Intended use/Application: Fragrance ingredient
Formulated concentrations were adjusted to allow for the stated water/impurity content (6.8%) of the test item.

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
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 24 years
Main Experiment: male, aged 24 years

MEDIA USED
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).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4-hour exposure to the test item with and 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, 115, 230, 460, 575, 690, 920 and 1840 µg/mL.
24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 57.5, 115, 230, 345, 460, 690 and 920 µg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity for all of the exposure groups in the preliminary toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO >99.9%

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The molecular weight of the test item was given as 184; therefore, the maximum dose level was 1840 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. The purity of the test item was 93.2% and was accounted for in the test item formulations.
The test item was immiscible in aqueous media at 18.4 mg/mL but was fully miscible in DMSO at 184 mg/mL in solubility checks performed in house.
Prior to each experiment, the test item was accurately weighed, formulated in DMSO 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 pH and osmolality readings are presented in the table found in "Any other information on materials and methods incl. tables" below.
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:

8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

The preliminary toxicity test was performed using all three of the exposure conditions as described below but using single cultures only.

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 0.1 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 0.1 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 0.1 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.

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, 7.19, 14.38, 28.75, 57.5, 115, 230, 460, 920 and 1840 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, 115, 230, 460, 575, 690, 920 and 1840 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, 115, 230, 460, 690, 920, 1150 and 1840 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, 57.5, 115, 230, 345, 460, 690 and 920 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 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.

Rationale for test conditions:

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.

Evaluation criteria:

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

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:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.19 to 1840 µg/mL. The maximum dose was the 10 mM concentration.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 460 µg/mL in the exposure groups in the absence of metabolic activation (S9) and at 1840 µg/mL in the exposure group in the presence of S9.
Hemolysis was observed following exposure to the test item at and above 57.5 µg/mL in all three exposure groups. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Additionally, a reduced cell pellet was observed at and above 230 µg/mL, 460 µg/mL and 920 µg/mL in the 4(20)-hour exposure groups, without and with S9, and the 24-hour continuous exposure group, respectively. A reduced cell pellet is indicative of toxicity to the cell population present and can indicate that maximum exposure has been reached.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 920 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24 hour continuous exposure was 460 µg/mL. The test item induced some evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity for all of the exposure groups.

Chromosome Aberration Test – Main Experiment
The dose levels of the controls and the test item are given in the section below (Any other information in results incl. tables).
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 920 µg/mL in the the 4(20)-hour exposure group in the absence of S9, up to 1150 µg/mL in the presence of S9 and up to 345 µg/mL in the 24-hour exposure group.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 920 µg/mL in the the 4(20)-hour exposure group in the absence of S9, at 1840 µg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 690 µg/mL in the 24-hour continuous exposure group. Haemolysis was observed in all dose levels tested in all three exposure groups. In addition, a reduced cell pellet was observed in the presence of S9 only at and above 1150 µg/mL.
The mitotic index data for the Main Experiment are given in the section below (Any other information in results incl. tables). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups.
In the 4(20)-hour exposure group in the absence of S9, 15% and 57% mitotic inhibition was achieved at 690 and 920 µg/mL, respectively. Above these dose levels, there were no metaphases present to analyze. Therefore, the maximum dose level selected for metaphase analysis was 920 µg/mL because this dose had achieved optimum toxicity as defined in the OECD 473 test guideline (55±5%).
In the 24-hour continuous exposure group, 21% and 57% mitotic inhibition was achieved at 115 and 230 µg/mL, respectively. Above these dose levels, there were either no or too few metaphases present to analyze. Consequently, the maximum dose level selected for metaphase analysis was 230 µg/mL because this dose had achieved optimum toxicity as defined in the OECD 473 test guideline (55±5%).
In the presence of S9, a very steep dose-related inhibition of mitotic index was observed as an inhibition of mitotic index of 17% was noted at 1150 µg/mL but there were no metaphase available to score at 1840 µg/mL. Therefore, the maximum dose level selected for metaphase analysis was 1150 µg/mL.

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.
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 the numbers of polyploid cells at any dose level in any of the 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 FRET13-0545(µg/mL)
4(20)-hour without S9	0*, 115, 230, 460, 575*, 690*, 920*, 1840, MMC0.4*
4(20)-hour with S9 (2%)	0*, 115, 230, 460, 690*, 920*, 1150*, 1840, CP2*
24-hour without S9	0*, 57.5*, 115*, 230*, 345, 460, 690, 920, MMC0.1*

*             = Dose levels selected for metaphase analysis

MMC   = Mitomycin C

CP         = Cyclophosphamide

The mitotic index data for the Main Experiment are given below:

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

Dose Level (mg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	11.40	12.15	11.78	100	7.35	7.40	7.38	100
115	- H	- H	-	-	- H	- H	-	-
230	- H	- H	-	-	- H	- H	-	-
460	11.25 H	11.50 H	11.38	97	- H	- H	-	-
575	8.75 H	10.50 H	9.63	82	NA	NA	NA	NA
690	10.00 H	9.95 H	9.98	85	8.40 H	7.10 H	7.75	105
920	5.25 H P	4.95 H P	5.10	43	8.40 H	7.90 H	8.15	110
1150	NA	NA	NA	NA	6.20 H R	6.05 H R	6.13	83
1840	NM H P	NM H P	-	-	NM H R P	NM H R P	-	-
MMC 0.4	4.70	4.75	4.73	40	NA	NA	NA	NA
CP 2	NA	NA	NA	NA	3.65	4.10	3.88	53

MMC            =Mitomycin C

CP                 =Cyclophosphamide

P                    =Precipitate

NA                =Not applicable

-                    =Not assessed for mitotic index

NM               =No metaphases suitable for scoring

H                   =Hemolysis

R                    = Reduced cell pellet

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	4.10	6.45	5.28	100
57.5	4.30 H	4.85 H	4.38	87
115	3.40 H	4.90 H	4.15	79
230	2.55 H	1.95 H	2.25	43
345	NM H	NM H	-	-
460	NM H	NM H	-	-
690	NM H P	NM H P	-	-
920	NM H P	NM H P	-	-
MMC 0.1	3.70	2.45	3.08	58

MMC            =Mitomycin C

P                    =Precipitate

NM               =No or too few metaphases suitable for scoring

H             =Hemolysis

Conclusions:

FRET 13-0545 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:

The test item, FRET 13-0545, was, considered to be non-clastogenic to human lymphocytes in vitro according to the OECD 473 Guideline the Mammalian Chromosome Aberration Test method (human peripheral blood lymphocytes).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

AMES

According to OECD 471, 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 μg/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 ranged between 0.5 to 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Up to eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally 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.

There were no toxicologically 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 (S9mix) in Experiment 1 (plate incorporation method) or Experiment 2 (pre incubation method).  A small, statistically significant increase in TA1535 revertant colony frequency was observed in the absence of S9-mix at 5000 µg/plate in the first mutation test.  This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted.  Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

In conclusion, FRET 13-0545 was considered to be non-mutagenic under the conditions of this test.

In vitro Chromosome Aberration

Following OECD 473, 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 on toxicity. The dose levels selected for the Main Test were as follows:

4(20)-hour without S9 -- 0, 115, 230, 460, 575, 690, 920, 1840

4(20)-hour with S9 (2%) -- 0, 115, 230, 460, 690, 920, 1150, 1840

24-hour without S9 -- 0, 57.5, 115, 230, 345, 460, 690, 920

All vehicle (dimethyl sulphoxide (DMSO)) 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 and to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that generally included a dose level that induced 55±5% mitotic inhibition.

The test item, FRET 13-0545 was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint
The results of the bacterial and chromosomal genotoxicity assays are reliable and adequate for covering this endpoint.

Short description of key information:
Ames test (OECD TG 471): negative
In vitro chromosome aberration test (OECD TG 473): negative

Endpoint Conclusion:No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the genotixicity assays, FRET 13 -0545 does not have to be classified for genotoxicity in accordance with Regulation (EC) No. 1272/2008.