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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2014

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2015

negative, in vitro micronucleus test (with and without S-9 activation), OECD TG 487, 2015

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:
19-11-2014 to 14-01-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test
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
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2012 ; signature: November 2012
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon (Salmonella strains)
tryptophan operon (E.coli strain)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone activated rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (pre-incubation method): range-finding test: 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.
TA 100 (- MA); TA 98, TA 1537 (+ MA); WP2 uvrA (+/- MA): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
TA 98, TA 1535, TA 1537 (- MA); TA 1535, TA 100 (+ MA): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration, Dimethyl sulphoxide was selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
WP2 uvrA: 2 µg/plate, TA 100: 3 µg/plate, TA 1535: 5 µg/plate (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537: 80 µg/plate (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
TA 98: 0.2 µg/plate (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 100: 1 µg/plate, TA 1535 & TA 1537: 2 µg/plate, WP2 uvrA: 10 µg/plate (with activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA 98: 5 µg/plate (with activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (pre-incubation)

DURATION
- Exposure duration: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were incubated at 37 ± 3 ºC 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). A number of manual counts were performed, predominantly due to colonies spreading, thus distorting the actual plate count.

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

NUMBER OF CELLS EVALUATED: the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were assessed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to the revertant colonies spreading slightly, thus distorting the actual plate count.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth and condition of background lawn.
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 is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in background lawn from 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in background lawn at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls (mean, n=3 plates)

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

Solvent control

77

92

16

9

22

30

22

18

6

7

1.5

73

78

14

12

20

23

17

21

6

6

5

72

87

18

12

16

26

26

15

11

8

15

68

81

9

9

19

28

23

19

12

11

50

79

86

12

11

24

27

17

20

13

8

150

61

78*

11*

12*

22

22

19*

19

5*

10

500

48*

76*

11*

0**

14*

23*

9*

23*

7*

4*

1500

0**

42***

0**

0***

0**

21*

0***

8**

0***

5*

5000

0***

0***

0***

0***

0***

14**

0***

0***

0***

0***

Positive control

519

496

2242

128

679

131

195

115

639

272

* Sparse bacterial background lawn

** Very weak bacterial background lawn

*** Toxic, no bacterial background lawn

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls (mean, n=3 plates)

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

- MA

+ MA

Solvent control

72

69

20

11

13

23

20

16

12

9

0.15

NT

71

9

11

NT

NT

16

NT

6

NT

0.5

67

75

17

11

10

20

18

20

5

9

1.5

78

75

15

12

10

18

19

12

7

9

5

70

67

15

9

13

20

19

18

9

9

15

74

67

11

9

15

22

15

20

10

7

50

57*

78*

7*

8*

12

19

14*

17

9*

7

150

58*

56*

7**

7*

11

12

16*

13*

8*

8*

500

44**

NT

NT

NT

9*

15*

NT

14*

NT

5*

Positive control

794

649

120

167

431

232

159

226

150

216

NT not tested

* Sparse bacterial background lawn

** Very weak bacterial background lawn

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls): Experiment 1 and 2, respectively

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

 

TA1535

 

WP2urvA

 

TA98

 

TA1537

 

82

 

12

 

20

 

27

 

13

 

86

(88)

8

(9)

19

(20)

13

(12)

11

(12)

95

 

8

 

21

 

27

 

13

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

 

TA1535

 

WP2urvA

 

TA98

 

TA1537

 

88

 

13

 

20

 

16

 

11

 

74

(78)

9

(11)

16

(18)

18

(15)

33

(8)

73

 

12

 

19

 

10

 

11

 

 

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14 and US EPA OCSPP harmonized guideline for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the 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 using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). Seven test item dose levels 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 dose range was amended following the results of Experiment 1 and ranged between 0.15 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. 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. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix, initially from 150 μg/plate (TA1535, TA98 and TA1537) and 500 μg/plate (TA100 and WP2uvrA). In the presence of S9-mix, weakened lawns were noted from 150 μg/plate (TA100 and TA1535) and 500 μg/plate (TA98, TA1537 and WP2uvrA). Consequently, the toxic limit was employed in the second mutation test. Visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 50 μg/plate in the absence of S9-mix and from 50 μg/plate (TA100 and TA1535) and 150 μg/plate (TA98 and TA1537) in the presence of S9-mix. Toxicity was also noted to E.coli strain WP2uvrA at 500 μg/plate in both the absence and presence of S9-mix. 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 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). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 50 μg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. The individual revertant counts at the statistically significant level were within the historical vehicle control range for the strain and the maximum increase was only two-fold the concurrent vehicle control. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-01-2014 to 10-07-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2014; signature: May 2014
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: 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 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. Further details on the donors is available in the full study report.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction: PB/βNF S9 20/10/13 and PB/βNF S9 15/12/13
Test concentrations with justification for top dose:
The maximum dose level was 1780 µg/mL, the 10 mM concentration, the maximum recommended dose level. 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) within the 0 to 1780 μg/mL range (full results recorded in the full study report).

I. Preliminary toxicity test: 0 (control) , , 7.0, 13.91, 27.81, 55.63, 111.25, 222.5, 445, 890 and 1780 μg/mL.
Within three exposure groups:
i) 4-hours 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-hours 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.

II. Main Test
Experiment 1:
4(20)-hour without S9: 0*, 60*, 90*, 180*, 240, 300, 360 μg/mL, MMC 0.4* μg/mL
4(20)-hour with S9 2%: 0*, 60*, 90*, 180*, 240*, 360, 450 μg/mL , CP 5* μg/mL
Experiment 2:
24-hour without S9: 0*, 15*, 30, 45*, 60*, 90*, 120 μg/mL MMC 0.2* μg/mL
4(20)-hour with S9 1%: 0*, 60*, 90*, 180*, 240, 360 μg/mL , CP 5* μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was miscible in both dimethyl sulphoxide and acetone at 178 mg/mL in solubility
checks performed in-house. Dimethyl sulphoxide was chosen as the preferred solvent vehicle. Prior to the start of each experiment, the test item was accurately weighed, formulated in dimethyl sulphoxide and serial dilutions prepared. The maximum dose level (determined prior to the test based on molecular weight) was 1780 µg/mL, which was calculated to be equivalent to 10mM, the maximum recommended dose level. 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) within the 0 to 1780 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
-MA: MMC 0.4 (experiment 1) and 0.2 (experiment 2) µg/ml and +MA: CP +MA, 5 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: Other:
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; 0.65 - 0.75 mL heparinized whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
I. With Metabolic Activation (S9) Treatment:
- 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 (100 μL) 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 of the Main Experiment. In Experiment 2, 1 mL of 10 % S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL. 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.

II. Without Metabolic Activation (S9) Treatment:
- 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 (100 μL) 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 at approximately 37 ºC in 5 % CO2 in humidified air. In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 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.

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: 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.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
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.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Evaluation criteria:
Positive response criteria
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) Either a concentration related or a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control is observed. Increases in only one dose level are assessed on a case-by case-basis.

Negative response criteria
A test item can be classified as non-genotoxic if:
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 and there is no concentration-related increase at any dose level.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.

Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. 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.
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. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
MI = 79% -S9 (180 µg/ml), 81 +S9 (240 µg/ml). Haemolysis occurred at higher concentrations. Steep toxicity curve observed, making concentrations to reach optimum toxicity difficult to achieve.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: here was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the Cell Growth Inhibition Test, a precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 445 μg/mL, in the exposure groups in the absence of metabolic activation (S9-mix) and at and above 890 μg/mL in the exposure group in the presence of S9-mix. Haemolysis was observed following exposure to the test item at and above 55.63 μg/mL in the 4(20)-hour exposure. In Experiment 1: Within the scored groups (up to 180 and 240 μg/mL, respectively) no precipitate was observed at the end of exposure, however, haemolysis was observed at and above 60 μg/mL and 90 μg/mL in the absence and presence of S9-mix, respectively. In Experiment 2: Within the scored groups (up to 90 and 180 μg/mL, respectively) no precipitate of the test item was observed at the end of exposure in either exposure group. However, haemolysis was observed at the end of exposure at and above 60 μg/mL in both exposure groups.
- Other confounding effects: In the preliminary test and the main test: Haemolysis was observed as described above. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1780 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).

ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Results of chromosome aberration study in Experiment 1 and 2

Concentration (µg/ml)

Total number and percentages of cells showing structural chromosome aberrations (%)

Mitotic index (%)

Number and percentages of cells showing numerical aberrations (%)

Polyploids

Others

Experiment 1: 4 hr treatment -MA

Solvent control

0 (0.0)

100

0

0

60

0 (0.0)

90

0

0

90

0 (0.0)

106

0

0

180

0 (0.0)

79

0

0

Positive control

33*** (33.0)

60

0

0

Experiment 1: 4 hr treatment +MA

Solvent control

0 (0.0)

100

0

0

90

0 (0.0)

99

0

0

180

3 (1.5)

102

0

0

240

2 (1.0)

81

1

0

Positive control

28*** (18.7)

25

0

0

Experiment 2: 24 hr treatment -MA

Solvent control

2 (1.0)

100

0

0

45

2 (1.0)

76

1

0

60

3 (1.5)

65

0

0

90

6 (3.0)

45

0

0

Positive control

32*** (32.0)

37

0

0

Experiment 2: 24 hr treatment +MA

Solvent control

0 (0.0)

100

0

0

60

0 (0.0)

62

0

0

90

0 (0.0)

78

0

0

180

0 (0.0)

72

0

0

Positive control

26*** (13.0)

28

0

0

*** = p<0.001

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The study was performed to the requirements of OECD TG 473, EU Method B.10 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. 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. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours 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 and a 4 hours exposure in the absence of metabolic activation (S9-mix) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9-mix was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on cytotoxicity. The dose levels selected for the Main Test Experiment 1 were as follows: 4(20)-hour with S9-Mix (2%): 0, 60, 90, 180, 240, 360 μg/mL and without S9-Mix: 0, 60, 90, 180, 240, 360, 450 μg/mL, respectively. In Experiment 2 the dose levels were: 24-hour without S9: 0, 60, 90, 180, 240, 300, 360 μg/mL and 4(20)-hour with S9 (1%): 0, 60, 90, 180, 240, 360 μg/mL, respectively. All vehicle (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 indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was cytotoxic but did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure groups in the absence and presence of S9, or the 4(20)-hour exposure group in the absence of S9. It was considered that the test item had been adequately tested since it was tested to cytotoxic dose levels. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-09-26 to 2015-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: March 2014 ; signature: May 2014
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM)
- Cells obtained from healthy non-smoking human volunteers who had not knowingly either been exposed to high levels of radiation or hazardous chemicals, or had a viral infection.
- The cell-cycle time was determined using BrdU (bromodeoxyuridine) and the average generation time (AGT) calculated. Mean AGT for regular donors – approximately 16 hours.

Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B: prior to cell harvest at final concentration of 4.5 μg/mL for 28 hours
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphthoflavone induced rat liver S9; S9 Microsomal fraction: PB/βNF S9 27/07/14, 12/10/14 and 23/11/14
Test concentrations with justification for top dose:
- Preliminary toxicity test: The dose range of test item used was 6.96 to 1783 μg/mL.
- Experiment I: 60, 90, 120, 150, 180, 210, 240 and 300 μg/mL, with and without metabolic activation
The exposure groups of Experiment 1 were initially performed with a dose range of 55.5 μg/mL to 444 μg/mL but due to the toxicity being greater than that seen in the Preliminary toxicity test and insufficient dose levels being available for scoring, the Experiment was repeated with the revised dose range above.
- Experiment II: 27.75, 55.5, 111, 148, 185 and 222 μg/mL, without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in DMSO up to 178.3 mg/mL, solubility checks were performed in-house
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
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
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: demecolcine
Remarks:
without metabolic activation
Details on test system and experimental conditions:
ACTIVATION: 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%.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I: 4 hours, with and without metabolic activation
Experiment II: 24 hours, without metabolic activation
- Expression time (cells in growth medium):
Experiment I: 28 hours with Cytochalasin B, with and without metabolic activation
Experiment II: 28 hours with Cytochalasin B, without metabolic activation

CYTOKINESIS BLOCK: Cytochalasin B (4.5 μg/mL)
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium

NUMBER OF REPLICATIONS: duplicate cell cultures (A and B) for each dose level

NUMBER OF CELLS EVALUATED: 500 cells per culture


CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, nonrefractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
Evaluation criteria:
The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the range of the laboratory historical control data. The frequency of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered valid.
All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
Statistics:
A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 210 μg/mL without S9 4 -hour exposure; at 300 μg/mL with S9, 4-hour exposure
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant change on pH was observed
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Precipitation: no precipitation of the test material was observed at any of the test concentrations

RANGE-FINDING/SCREENING STUDIES:
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 891.5 μg/mL and 445.75μg/L, in the 4-hour exposure group in the absence and presence of S9 respectively and at and above 445.75 μg/mL in the 24-hour continuous exposure group. Haemolysis was observed following exposure to the test item at and above 111.44 μg/mL in the 4-hour exposure groups in the presence and absence of S9 and in the 24-hour continuous exposure group.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See tables 1, 2 and 3.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: A minimum of 500 cells per culture were scored.
- Indication whether binucleate or mononucleate where appropriate: See table 1, 2 and 3. The vehicle control cultures had frequencies of binucleate cells with micronuclei within the expected range. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See tables 1, 2 and 3. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
- Negative (solvent/vehicle) historical control data: The frequency of spontaneous binucleate cells with micronuclei in the vehicle control cultures was within the range of the laboratory historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI cytokinesis-block method (see tables 1, 2 and 3).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Results, experiment I, 4 -hour exposure without metabolic activation

4 - hour exposure without S9

Dose level (μg/mL)

Replicate

Nucleate cells/ 500 cells

CBPI

Mean CBPI

Cytotoxicity (% Control CBPI)

Mono

Bi

Multi

0

A

211

256

33

1.64

1.63

100

B

223

245

32

1.62

60

A

-

-

-

-

-

-

B

-

-

-

-

90

A

255

232

13

1.52

1.40

63

B

363

135

2

1.28

120

A

288

197

15

1.45

1.47

75

B

262

232

6

1.49

150

A

295

199

6

1.42

1.40

63

B

317

183

0

1.37

180

A

302

192

6

1.41

1.37

59

B

341

154

5

1.33

210 +

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

240 +

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

300 +

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

MMC 0.2

A

321

176

3

1.36

1.40

63

B

285

211

4

1.44

Table 2. Results, experiment I, 4 -hour exposure with metabolic activation

4 - hour exposure with S9

Dose level (μg/mL)

Replicate

Nucleate cells/ 500 cells

CBPI

Mean CBPI

Cytotoxicity (% Control CBPI)

Mono

Bi

Multi

0

A

57

375

68

2.02

1.93

100

B

123

332

45

1.84

60

A

-

-

-

-

-

-

B

-

-

-

-

90

A

-

-

-

-

-

-

B

-

-

-

-

120

A

-

-

-

-

-

-

B

-

-

-

-

150

A

102

346

52

1.90

1.86

92

B

129

336

35

1.81

180

A

76

363

61

1.97

1.91

98

B

114

347

39

1.85

210

A

184

284

32

1.70

1.82

88

B

72

384

44

1.94

240

A

262

230

8

1.49

1.51

55

B

244

249

7

1.53

300 +

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

CP 5

A

312

185

3

1.38

1.47

50

B

241

243

16

1.55

Table 3. Results, experiment II, 24 -hour exposure without metabolic activation

24 - hour exposure without S9

Dose level (μg/mL)

Replicate

Nucleate cells/ 500 cells

CBPI

Mean CBPI

Cytotoxicity (% Control CBPI)

Mono

Bi

Multi

0

A

152

309

39

1.77

1.78

100

B

147

310

43

1.79

27.75

A

174

292

34

1.72

1.73

94

B

162

305

33

1.74

55.5

A

193

277

30

1.67

1.67

85

B

191

286

23

1.66

111

A

320

178

2

1.36

1.39

50

B

295

199

6

1.42

148

A

424

75

1

1.15

1.16

20

B

419

81

0

1.16

185

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

222

A

NB

NB

NB

NB

NB

NB

B

NB

NB

NB

NB

DC 0.075

A

238

199

63

1.65

1.59

76

B

288

160

52

1.53

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

- = Not selected for scoring

NB = No binucleate cells or insufficient binucleate cells for scoring

H = Haemolysis observed at the end of exposure

+ = Reduced cell pellet observed at the end of exposure

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

The study was performed to the requirements of OECD TG 487 : guidelines under GLP conditions to assess within the in vitro cell micronucleus assay the clastogenic and aneugenic potential of the test item to the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2% final concentration). Experiment 2, used a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of Cytochalasin B. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. The dose levels were as follows: 4-hour without S9-Mix and 4-hour with S9-Mix (2%): 60, 90, 120, 150, 180, 210, 240, 300 μg/mL. In a second experiment the dose levels were 4-hour without S9: 37.75, 55.5, 111, 148, 185, 222 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of binucleate cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately a 50% reduction in CBPI in all three exposure groups. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD TG 471, 2014 -The study was performed to the requirements of OECD Guideline 471, EU Method B13/14 and US EPA OCSPP harmonized guideline for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the 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 using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and ranged between 0.15 and 500 μg/plate, depending on bacterial strain type and presence or absence of metabolic activation (S9-mix). Seven test item dose levels 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 dose range was amended following the results of Experiment 1 and ranged between 0.15 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. 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. 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 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). A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 50 μg/plate in the range-finding test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. The individual revertant counts at the statistically significant level were within the historical vehicle control range for the strain and the maximum increase was only two-fold the concurrent vehicle control. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

 

OECD TG 473, CAT in vitro: 2015 -The study was performed to the requirements of OECD TG 473, EU Method B.10 and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. 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. Four treatment conditions were used for the study; i.e. in Experiment 1, 4 hours 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 and a 4 hours exposure in the absence of metabolic activation (S9-mix) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9-mix was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on cytotoxicity. The dose levels selected for the Main Test Experiment 1 were as follows: 4(20)-hour with S9-Mix (2%): 0, 60, 90, 180, 240, 360 μg/mL and without S9-Mix: 0, 60, 90, 180, 240, 360, 450 μg/mL, respectively. In Experiment 2 the dose levels were: 24-hour without S9: 0, 60, 90, 180, 240, 300, 360 μg/mL and 4(20)-hour with S9 (1%): 0, 60, 90, 180, 240, 360 μg/mL, respectively. All vehicle (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 indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was cytotoxic but did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure groups in the absence and presence of S9, or the 4(20)-hour exposure group in the absence of S9. It was considered that the test item had been adequately tested since it was tested to cytotoxic dose levels. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

 

OECD TG 487, MN in vitro: 2015 - The study was performed to the requirements of OECD TG 487 : guidelines under GLP conditions to assess within the in vitro cell micronucleus assay the clastogenic and aneugenic potential of the test item to the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2% final concentration). Experiment 2, used a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of Cytochalasin B. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. The dose levels were as follows: 4-hour without S9-Mix and 4-hour with S9-Mix (2%): 60, 90, 120, 150, 180, 210, 240, 300 μg/mL. In a second experiment the dose levels were 4-hour without S9: 37.75, 55.5, 111, 148, 185, 222 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of binucleate cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately a 50% reduction in CBPI in all three exposure groups. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity