Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity potential of the substance was investigated in an in vitro Ames test according to OECD 471, in an in vitro chromosome aberration test according to OECD 473 and in an in vitro gene mutation test in mammalian cells according to OECD 490. The substance was tested negative with and without metabolic activation in all three available in vitro tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-12-2016 to 24-01-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1 Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 Pre-Incubation Method: 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
dimethyl sulphoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Positive controls:
yes
Positive control substance:
9-aminoacridine
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
- Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
8 concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/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 containing. 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 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.

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
6 concentrations of the test item (15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed.

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 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 and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

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:
test item is considered non-mutagenic (negative) in the test system if the blew criteria are not met.
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)).
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05)
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
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
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
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
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:
After employing the pre-incubation modification in the second mutation test a greasy looking precipitate was noted at and above 1500 µg/plate, this observation did not interfere with the scoring of revertant colonies.
A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 500 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility
Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-07-2017 to 08-12-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 29 July 2016
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines
Version / remarks:
31 March 2011
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Remarks:
human
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.

The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: male, aged 31 years
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The results of an experimental pre-test were used to select the concentrations for the chromosome aberration assays.
For concentrations, see section "Details on test systems and conditions"
Vehicle / solvent:
dimethyl sufoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
- 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).

- 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%.

- Duplicate lymphocyte cultures (A and B) were established for each dose level

- Preincubation of cultures : appr. 48 hours at 37°C

-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.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.

-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, 62.5, 125, 250, 500, 7500 and 1000 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, 31.25, 62.5, 125, 250, 375 and 500 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, 31.25, 62.5, 125, 187.5, 250 and 375 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.

- 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.

- 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. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. A dose-related increase in polyploid cells are reported separately. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
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 except for the negative control and dose level 187.5 µg/mL in the 24-hour continuous exposure group where 600 cells were analyzed to confirm a response observed in the first 300
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.
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 7.81 to 2000 µg/mL. The maximum dose was the maximum recommended dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at 2000 µg/mL, in the 4(20)-hour exposure group in the absence of metabolic activation (S9) in the continuous exposure group. In the 4(20)-hour exposure group in the presence of metabolic activation (S9) precipitate was observed at and above 125 µg/mL.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 500 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 250 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 the lowest precipitating dose level and toxicity in the presence of S9-mix and on toxicity alone in the absence of S9-mix.

Chromosome Aberration Test – Main Experiment:
- The dose levels of the controls and the test item are given in the table below:

- The qualitative assessment of the slides determined that the precipitate and predicated toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to maximum dose level in all three exposure groups, albeit in numbers too low to consider analyzing.

- In the 24-hour continuous exposure group, a small increase in the frequency of cells with aberrations was noted at 187.5 µg/mL, predominantly in the ‘B’ culture. No increases in the frequency of cells with aberrations were noted in the 4(20)-hour exposure groups in the presence and absence of metabolic activation (S9) even though all exposure groups had meet the OECD 473 acceptability criteria for toxicity . Therefore, the duplicate slides of the negative control and for dose level 187.5 µg/mL were assessed and the results added to the original 300 cells in an effort to resolve or clarify the result. The assessment of 600 cells from these dose groups concluded that the test item was not producing a statistically significant increase in cells with aberrations present.
Additionally, a statistically significant increase in the numbers of polyploid cells at 250 and 500 µ/mL in the 4(20)-hour exposure group in the absence of S9-mix only was observed. Polyploidy is considered to be the effect of an interruption of the mitotic apparatus, particularly disturbances to spindle formation, during mitosis. Although it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells to the study, the endpoint is the collation of structural chromosome aberrations and the overriding purpose of this study.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH of test item preparation: no significant changes
- Effects of osmolality of test item preparation: no increase by more than 50mOsm
- Water solubility: test item insoluble in aqueous media at 20 mg/mL
- Precipitation: Precipitate observations were made at the end of exposure in the cultures and was noted at and above 375 µg/mL in the 4(20)-hour exposure group in the presence of S9 only. No precipitate was observed in the absence of S9-mix
- Definition of acceptable cells for analysis: see above ("Evaluation criteria")


HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index; a dose-related inhibition of mitotic index was observed in all three exposure groups.

 Group  Final concentration of ELA162 (µg/mL)
 4(20)-hour without S9 0*, 0, 62.5, 125*, 250*, 500*, 750, 1000, MMC 0.2* 
 4(20)-hour with S9 (2%) 0*, 31.25, 62.5, 125*, 250*, 375*, 500, CP 2* 
 24-hour without S9 0*, 31.25, 62.5, 125*, 187.5*, 250*, 375, MMC 0.2* 

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:
The test item 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 was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-05 to 2018-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK +/- locus of L5178Y
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Additional strain / cell type characteristics:
other: TK +/- 3.7.2c
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Main Test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 187.5, 250 µg/L
The dose range was selected following the results (toxicity and precipitate) of a preliminary toxicity test. The maximum dose level used in the main test was limited by a combination of test item induced toxicity and precipitate.
Preliminary test: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item is insoluble in aqueous media at 20mg/mL but was soluble in DMSO at 200mg/mL in solubility checks performed in-house.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 1x10^6 cells/mL in 10mL aliquotes in R10 medium (4h-exposure); 0.3x10^6 cells/mL in 10mL aliquotes in R10 medium (24h-exposure);

DURATION
- Preincubation period: none
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 - 12 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Small and large colony evaluation
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in Mutation Frequency (MF) above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: yes
- Definition of acceptable cells for analysis: Dose levels that have Relative Total Growth (RTG) survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance


RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups (4 h- exposure (-S9); 4h-exposure (+S9); 24h-exposure (-S9)) when compared to the concurrent vehicle control groups. The toxicity curve was steep in all three exposure groups. Precipitate of the test item was observed at and above 125 µg/mL in both 4-hour exposure groups. Precipitate of the test item was observed and above 250 µg/mL in the 24hour exposure group




HISTORICAL CONTROL DATA (with means and standard deviation)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Table 1: Summary of results - Main experiment

Treatment

4-hours-S-9 Treatment

Treatment

4-hours+S-9

Treatment

24-hours-S-9

(µg/mL)

(µg/mL)

(µg/mL)

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

100

1

166.66

0

100

1

148.31

0

100

1

132.03

3.91 Ø

92

 

 

3.91

104

0.91

153.47

3.91

72

 

 

7.81

95

1.03

141.48

7.81

105

1.06

136.45

7.81

110

0.82

206.24

15.63

95

0.98

154.19

15.63

101

0.98

140.22

15.63

99

0.79

154.03

31.25

95

0.97

170.21

31.25

93

0.84

144.53

31.25

88

0.81

135.81

62.5

82

0.88

163.51

62.5

81

0.79

153.59

62.5

71

0.76

167.3

125

79

0.73

181.94

125

77

0.69

161.57

125

44

0.41

241.33

187.5

54

0.54

187.66

187.5 Ø

52

 

 

187.5 Ø

20

0.27

227.51

250 Ø

26

 

 

250 Ø

36

 

 

250 Ø

7

 

 

MF threshold for a positive response= 292.66

 MF threshold for a positive response= 274.31

 MF threshold for a positive response= 258.03

EMS

 

 

 

CP

 

 

 

EMS

 

 

 

400

83

0.72

1319.01

1.5

87

0.6

1428.35

150

59

0.44

1274.43

Ø = Not plated surplus to requirements

RTG = Relative Total Growth

%RSG = Relative Suspension Growth

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure

Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be nonmutagenic in this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data the substance will not be classified.