Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test performed according to OECD 471, FRET 14 -0383 was considered to be non-mutagenic under the conditions of this test

In a chromosome aberration test performed according to OECD 473, FRET 14 -0383 was considered to be non-clastogenic to human lymphocytes in vitro.

Link to relevant study records

Referenceopen allclose all

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 25 January 2017 and 02 June 2017
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)
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:
Physical state/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 ºC in the dark
The purity of the test item was accounted for in test item formulation
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: Whole blood cultures from the following donors: Preliminary Toxicity Test: male, aged 25 years Main Experiment: female, aged 24 years
Details on mammalian cell type (if applicable):
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.

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
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration
Test concentrations with justification for top dose:
The molecular weight of the test item was given as 163, therefore, the maximum dose level was 1630 µg/mL, 10mM, the maximum recommended dose level.
Preliminary toxicity test: 6.37 to 1630 μg/mL (with and without S-9)
Main study: All groups: 0, 6.25, 12.5, 25, 37.5, 50, 75 and 100 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
The test item was insoluble in MEM at 16.3 mg/mL and in Acetone at 163 mg/mL but was soluble in DMSO at 163.0 mg/mL in solubility checks performed in house.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.4 µg/mL for 4-hour exposure and 0.2 µg/mL for 24-hour exposure
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
PREPARATION OF CULTURES: 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).

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 or 24 hours, 37 ºC
- Fixation time (start of exposure up to harvest of cells): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/mL) 2.5 hours
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED:
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.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- 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:
Criteria 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. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• 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.

Providing that the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, under any of the experimental conditions:
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.

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.
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.
Species / strain:
lymphocytes: human
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 6.37 to 1630 µg/mL. The maximum dose was the 10 mM concentration.
A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 407.5 µg/mL in the absence of S9 and at and above 203.75 µg/mL in the presence of S9, in the 4(20)-hour exposure groups.

Greasy/oily precipitate was also observed in the parallel blood-free cultures at the end of the exposure, at and above 203.75 µg/mL in the absence of S9 and at and above 815 µg/mL in the presence of S9, in the 4(20)-hour exposure groups. In the continuous exposure group cloudy precipitate was observed at and above 101.88 µg/mL.
Haemolysis was observed following exposure to the test item at and above 25.47µg/mL in the 4(20)-hour exposure groups and the 24-hour continuous exposure group. Haemolysis 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 50.94 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 also 50.94 µg/mL. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 100 µg/mL for all three exposure groups.

Chromosome Aberration Test – Main Experiment

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 in all three exposure groups. In the presence of metabolic activation (S9) and the continuous exposure group, the maximum dose level of the test item with metaphases suitable for scoring was 75 µg/mL. In the absence of metabolic activation (S9) the maximum dose level of the test item with metaphases suitable for scoring was 50 µg/mL.
Precipitate observations were made at the end of exposure and no precipitate was observed. Haemolysis was observed in the presence of metabolic activation (S9) and the continuous exposure group at and above 75 µg/mL and in the absence of metabolic activation (S9) at and above 50 µg/mL.
They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 55% mitotic inhibition was achieved at 50 µg/mL. In the presence of S9, 41% mitotic inhibition was achieved at 75 µg/mL. An inhibition of mitotic index of 43% was noted at 75 µg/mL in the 24-hour continuous exposure group.
The maximum dose level selected for metaphase analysis was 50 µg/mL in the 4(20)-hour exposure group in the absence of S9 and 75 µg/mL in the 4(20)-hour exposure group in the presence of S9 and 24-hour continuous exposure group.

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 all of the exposure groups.

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration ofFRET 14-0383(µg/mL)

4(20)-hour without S9

0*, 6.25, 12.5*, 25*, 37.5*, 50*, 75, 100, MMC0.4*

4(20)-hour with S9 (2%)

0*, 6.25, 12.5, 25*, 37.5*, 50*, 75*, 100, CP2*

24-hour without S9

0*, 6.25, 12.5, 25*, 37.5*, 50*, 75*, 100, MMC0.2*

MMC = Mitomycin C

*  = Dose levels selected for metaphase analysis

CP = Cyclophosphamide

 

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

6.50

100

8.45

100

8.45

100

6.37

-

-

-

-

-

-

12.73

9.75

150

7.05

83

7.25

86

25.47

7.90 H

122

5.15 H

61

6.45 H

76

50.94

2.25 H

35

5.60 H

66

3.90 H

46

101.88

NM H

NM

NM H

NM

NM H G/O

NM

203.75

NM H G/O

NM

NM H C

NM

NM H G/O

NM

407.5

NM H G/O C

NM

NM H C

NM

NM H G/O

NM

815

NM H G/O C

NM

NM H G/O C

NM

NM H G/O

NM

1630

NM H G/O C

NM

NM H G/O C

NM

NM H G/O

NM

-      = Not assessed for mitotic index

NM   = No metaphases or insufficient metaphases suitable for scoring

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

G/O= Greasy/oily precipitate observed at end of exposure period in blood-free cultures

C     = Cloudy precipitate observed at end of exposure period in blood-free cultures

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

9.45

6.90

8.18

100

7.80

9.20

8.50

100

6.25

-

-

-

-

-

-

-

-

12.5

8.20

9.45

8.83

108

-

-

-

-

25

7.50 H

9.90 H

8.70

106

6.85

8.40

7.63

90

37.5

8.00 H

8.90 H

8.45

103

9.55 H

9.85 H

9.70

114

50

2.50 H

4.90 H

3.70

45

9.20 H

7.00 H

8.10

95

75

NM H

NM H

NM

NM

5.20 H

4.80 H

5.00

59

100

NM H

NM H

NM

NM

NM H

NM H

NM

NM

MMC 0.4

2.00

2.25

2.13

26

NA

NA

NA

NA

CP 5

NA

NA

NA

NA

3.90

3.45

3.68

43

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysis

 

 


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

Dose Level

(µg/mL)

24-Hour Without S9

A

B

Mean

% of Control

0

4.50

6.05

5.28

100

6.25

-

-

-

-

12.5

-

-

-

-

25

6.70

4.50

5.60

106

37.5

3.80 H

5.80 H

4.80

91

50

4.00 H

3.50 H

3.75

71

75

2.20 H

3.80 H

3.00

57

100

NM H

NM H

NM

NM

MMC 0.2

1.45

2.85

2.15

41

MMC = Mitomycin C

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysis

 


Conclusions:
Interpretation of results (migrated information):
negative

Under the test conditions, FRET 14-0383 is not considered as clastogenic in human lymphocytes according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to FRET 14-0383 in DMSO at concentration range of 6.25 - 100 μg/mL, for 4 + 24 h (treatment + recovery) with metabolic activation (2% S-9), and for 4 + 20 h or 24 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test.

 

In the main test, all experiments were performed at concentrations up to 100 µg/mL without S-9 and with S-9 and the following concentrations were selected for analysis:

4(20)-hour without S9: 0, 12.5, 25, 37.5, and 50 μg/mL

4(20)-hour with S9 (2%): 0, 25, 37.5, 50 and 75 µg/ml

24-hour without S9: 0, 25, 37.5, 50 and 75 µg/ml

 

Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (Mitomycin C at 0.4 and 0.2 µg/mL (4 and 24 hr treatment groups respectively) without S-9 and cyclophosphamide at 2 µg/mL with S-9) induced the appropriate response. Treatment of cells with FRET 14-0383 in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed.

 

Under the test conditions, FRET 14-0383 is not considered as clastogenic in human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 20 January 2017 and 24 February 2017
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:
EC No. 440/2008 of 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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: FRET 14-0383
Chemical name: 4,7-METHANO-1H-INDENE, OCTAHYDRO-1-(METHOXYMETHYLENE)- MIXTURE
Physical state/Appearance: Clear colourless liquid
Batch: GM435-17
Purity: 95.2%
Expiry Date: 01 January 2019
Storage Conditions: Approximately 4°C in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (4.8%) of the test item
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan 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 for all stains, using the direct plate incorporation method:
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 2:
As the result of experiment 1 was negative the following dose levels were used for experiment 2:
Salmonella strains TA100 (with and without S9-mix) and TA1535 and TA98 (without S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains TA1537 (with and without S9-mix) and TA1535 and TA98 (with S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both 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
- Justification for choice of solvent: the test substance was found to be soluble in DMSO
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:
Experimental Design and Study Conduct
Test Item Preparation and Analysis
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 selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 05 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (4.8%) of the test item. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see above) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
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).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100 (with and without S9-mix) and TA1535 and TA98 (without S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains TA1537 (with and without S9-mix) and TA1535 and TA98 (with S9 mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
WP2uvrA- (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both 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.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously (see above) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3”C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
Incubation and Scoring
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). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count
Evaluation criteria:
Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination

Evaluation Criteria
Any, one, or all of the following criteria 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.

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, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle 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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In all strains no toxicity was observed at 5000 µg/plate, both in the absence and presence of S9 in the dose range finder, Experiment 1.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In all strains no toxicity was observed at the higher dose (5000 µg/plate), both in the absence and/or presence of S9

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) are presented in Table 1 and were considered to be acceptable.  These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.  

A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.  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 150 µg/plate in the absence of S9 mix and 500 µg/plate in the presence of S9-mix.  For Experiment 2, the test item was tested up to either the toxic limit or the maximum recommended dose level of 5000 µg/plate, depending on bacterial strain type.  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 15 µg/plate (TA1535), 50 µg/plate (TA100, TA98 and TA1537) and 150 µg/plate (WP2uvrA).  In the presence S9-mix, weakened bacterial background lawns were noted to all of the Salmonella strains from 500 µg/plate and at 5000 µg/plate to WP2uvrA.  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 5 µg/plate in the first mutation test.  This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.  Furthermore, the individual revertant colony counts at 5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

The vehicle (dimethyl sulphoxide) 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:
FRET 14-0383 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 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 slightly, following the results of Experiment 1 and ranged between 0.15 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.  Eight 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 (dimethyl sulphoxide) 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.  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 150 µg/plate in the absence of S9 mix and 500 µg/plate in the presence of S9-mix.  For Experiment 2, the test item was tested up to either the toxic limit or the maximum recommended dose level of 5000 µg/plate, depending on bacterial strain type.  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 15 µg/plate (TA1535), 50 µg/plate (TA100, TA98 and TA1537) and 150 µg/plate (WP2uvrA).  In the presence S9-mix, weakened bacterial background lawns were noted to all of the Salmonella strains from 500 µg/plate and at 5000 µg/plate to WP2uvrA.  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 5 µg/plate in the first mutation test.  This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.  Furthermore, the individual revertant colony counts at 5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control.

Conclusion

FRET 14-0383 was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames test (OECD 471):

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains (>=250 ug/pl). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

FRET 14-0383 was considered to be non-mutagenic under the conditions of this test.

In vitro chromosome aberration assay (OECD 473):

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to FRET 14-0383 in DMSO at concentration range of 6.25 - 100 μg/mL, for 4 + 24 h (treatment + recovery) with metabolic activation (2% S-9), and for 4 + 20 h or 24 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test.

Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (Mitomycin C at 0.4 and 0.2 µg/mL (4 and 24 hr treatment groups respectively) without S-9 and cyclophosphamide at 2 µg/mL with S-9) induced the appropriate response. Treatment of cells with FRET 14-0383 in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed.

Under the test conditions, FRET 14-0383 is not considered as clastogenic in human lymphocytes in vitro.


Justification for classification or non-classification

Based on the results of the Ames test and in vitro chromosome aberration test the substance does not have to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008 and its amendments.