Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 97, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

In vitro mammalian chromosome abberation test (OECD 473): negative in V79 cells with and without metabolic activation

In vitro gene mutation in mammalian cells (OECD 476): negative in V79 cells with and without metabolic activation

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:
16 Feb - 09 Mar 2018
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:
adopted in 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovenska Republika
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (for S. typhimurium strains), trp operon (for E. coli strain)
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor
Test concentrations with justification for top dose:
Range-finding study: 50, 150, 300, 500, 1500 and 5000 µg/plate in TA 97 and TA 100 without metabolic activation

First main experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second main experiment: 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: DMSO was chosen due to the insolubility of the test substance in water. The vehicle used did not affect the spontaneous mutation level and it is recommended for use in this test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Range Finding Test and first main experiment); preincubation (second main experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: The bacterial growth was controlled in order to determine toxicity of the test substance.
Evaluation criteria:
Acceptance criteria
The study is considered valid if all of the following criteria are met:
- tester strains fulfil the criteria for sensitivity to UV light
- tester strains exhibit sensitivity to crystal violet
- tester Salmonella strains TA97, TA98, TA100 strain exhibit resistance to ampicillin
- all tester Salmonella strains and E.coli WP 2 uvrA exhibit a characteristic number of spontaneous revertant colonies when plated. The mean should be within the range of historical control values or within the published historical range
- all tester strains exhibit at least a three-fold increase in mutagen-induced revertant colonies when plated with positive control chemicals

Evaluation criteria
Considering biological relevance the test substance is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2

The positive result indicates that the test substance induces mutations in Salmonella typhimurium or E.coli cells.

The test item for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the test conditions, the test substance does not produce mutations in Salmonella typhimurium or E.coli cells.
Statistics:
The mutation frequency at each dose concentration level of the test item was compared to the one observed in negative and positive controls. The statistical analysis was carried out using unpaired T-test.
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No inhibition of the bacterial growth was observed in the two tested Salmonella strains TA 97 and TA 100 in the concentration range 50 - 5000 µg/plate without metabolic activation. Therefore the same doses (except 300 µg/plate) were tested in all Salmonella strains (TA 97, TA 98, TA 100 and TA 1535) and in E. coli WP2 uvr A in the main tests, with and without metabolic activation.

HISTORICAL CONTROL DATA
The mean revertants for controls for Salmonella tester strains and E.coli WP2 uvr were within the range of historical control values (please refer to Table 3 in "Any other information on results incl. tables").

Table 1:Summary of test results (experiment 1; Plate Incorporation Method)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates ± SD)

Frameshift type

Base-pair substitution type

TA 97

TA98

TA100

TA1535

WP2 uvr A

Solvent control (DMSO)

124 ± 3

18 ± 2

145 ± 12

17 ± 3

26 ± 7

50

116 ± 3 *

17 ± 3

127 ± 3

16 ± 3

24 ± 5

150

119 ± 2

19 ± 3

130 ± 2

14 ± 1

32 ± 3

500

105 ± 5 *

18 ± 7

122 ± 18

15 ± 3

28 ± 9

1500

128 ± 9

17 ± 3

148 ± 19

17 ± 3

25 ± 4

5000

115 ± 8

18 ± 4

144 ± 7

18 ± 3

33 ± 5

Positive
controls (µg/plate)

9AA
(75)

2NF
(3)

SA
(1.5)

SA
(1.5)

4NQO
(5)

Mean No. of colonies/plate (average of 3 plates)

577 ± 38 *

432 ± 78 *

531 ± 28 *

448 ± 71 *

145 ± 8 *

+

Solvent control (DMSO)

152 ± 16

28 ± 9

133 ± 13

18 ± 5

39 ± 4

50

136 ± 16

28 ± 2

134 ± 6

22 ± 7

44 ± 3

150

149 ± 9

27 ± 1

124 ± 15

19 ± 3

43 ± 2

500

153 ± 11

26 ± 8

113 ± 21

15 ± 2

38 ± 6

1500

148 ± 4

26 ± 1

128 ± 7

17 ± 1

39 ± 8

5000

159 ± 9

28 ± 2

118 ± 7

17 ± 2

43 ± 2

Positive
controls
 

(µg/plate)

2AA
(5)

Mean No. of colonies/plate (average of 3 plates)

549 ± 57 *

159 ± 12 *

772 ± 25 *

211 ± 8 *

328 ± 17 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 2: Summary of test results (experiment 2; Preincubation Method)

 

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates ± SD)

Frameshift type

Base-pair substitution type

TA 97

TA98

TA100

TA1535

WP2 uvr A

Without S9 Mix

Solvent control (DMSO)

159 ± 10

18 ± 5

130 ± 17

14 ± 3

34 ± 8

15

145 ± 8

17 ± 7

129 ± 8

13 ± 3

28 ± 6

50

151 ± 2

13 ± 4

119 ± 12

16 ± 1

28 ± 1

150

151 ± 2

17 ± 5

116 ± 4

15 ± 2

30 ± 4

500

153 ± 10

16 ± 2

117 ± 12

15 ± 3

34 ± 4

1500

153 ± 12

13 ± 4

116 ± 17

14 ± 2

31 ± 4

5000

155 ± 4

15 ± 1

127 ± 7

12 ± 4

33 ± 6

Positive
controls (µg/plate)

9AA
(75)

2NF
(3)

SA
(1.5)

SA
(1.5)

4NQO
(5)

Mean No. of colonies/plate (average of 3 plates)

657 ± 60 *

400 ± 17

531 ± 28 *

448 ± 71 *

145 ± 8 *

2AA = 2-aminoanthracene

2NF = 2-nitro-fluorene

4NQO = 4-nitroquinoline-1-oxide

9AA = 9-aminoacridine

SA = sodium azide

SD = standard deviation

* = p < 0.05

Table 3: Historical negative and positive control values (2015 -2018)

S9 Mix   Revertants per plate
TA 100 TA 1535 TA 97 TA 98 WP2 uvr A
Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos
- Mean 194 698 23 624 163 1135 25 246 38 423
SD 35 170 8 170 30 527 7 141 12 270
Min 115 440 6 336 114 400 12 117 25 82
Max 272 1148 43 964 227 2776 43 680 68 912
+ Mean 180 1041 23 192 195 893 36 779 46 397
SD 29 443 8 130 36 369 12 438 10 145
Min 123 398 11 34 127 370 19 256 31 194
Max 231 2888 43 604 286 1616 85 1644 70 648

SD = standard deviation

Min = minimum value

Max = maximum value

Neg = negative control

Pos = positive control

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells tested up to limit concentration.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jun - 08 Oct 2018
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 in 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovak National Accreditation Service, Bratislava, Slovak Republic
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The master stock of V79 was obtained from ECACC (European Collection of Cell Cultures), UK (originally established by Ford and Yerganian in 1958)
- Number of passages: 3 to 6
- Methods for maintenance in cell culture if applicable: When the cells were growing well, subcultures were established in an appropriate number of Petri dishes. Subculturing was performed 2 - 3 times a week with the aid of trypsin (0.25 %)/EDTA solution and counted in suspension using Bürker’s chamber.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco´s Modified Eagle´s Medium (DMEM) with 4.5% glucose supplemented with L-gluamine, 100 units/mL Penicillin and 100 µg/mL Streptomycin. 10% fetal bovine serum was added to growth media.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
demecolcine (0.1 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with 20-methylcholanthrene
Test concentrations with justification for top dose:
3 h incubation (-S9 mix): 1.18, 2.35, 3.76, 4.23 and 4.70 µg/mL
3 h incubation (+S9 mix): 9.40, 11.28, 13.16, 14.10,15.04, 16.92, 17.86, 18.80, 21.15, 23.50, 25.85 and 28.20 µg/mL
21 h incubation (- S9 mix): 0.94, 3.29, 4.70, 6.11, 6.58, 7.05 and 7.52 µg/mL

The following concentrations were selected for scoring:
3 h incubation (-S9 mix): 1.18, 2.35 and 3.76 µg/mL
3 h incubation (+S9 mix): 18.80, 21.15 and 23.50 µg/mL
21 h incubation (- S9 mix): 3.29, 4.70 and 6.58 µg/mL

Concentrations selected for the chromosome aberration test were based on the results of the cytotoxicity range-finding experiment where concentrations of up to 65.8 µg/mL (maximum concentration) were tested.

Vehicle / solvent:
- Solvent used: ethanol with Tween 80 (10 mg/mL)
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:
METHOD OF APPLICATION: in suspension
- Cell density at seeding: 3.5 x 10E5 per Petri dish

DURATION
- Exposure duration: 3 h with and without metabolic activation and 21 h without metabolic activation
- Expression time (cells in growth medium): 18 h for 3 h exposure time, the 21 h exposure groups were continously exposed
- Fixation time (start of exposure up to harvest of cells): 21 h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (0.1 µg/mL)

STAIN (for cytogenetic assays): After air drying, the slides were coded and stained with Giemsa stain (3 % in distilled water) for 8 min.

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the demecolcine exposure, the media with dividing cells were transferred into labelled centrifuge tubes, the monolayer of cells was dissociated with 0.25 % trypsin and resuspended in the collected medium. An aliquot of this cell suspension was counted using Bürker`s chamber for determination of relative increase in cell counts (RICC). The number of cells per dish was calculated for each concentration, and percentages of RICC were calculated. The chosen samples based on cytotoxicity were then centrifuged (1000 rpm for 5 min). The culture medium was removed and approximately 8 mL of hypotonic potassium chloride was added to swell the cells. After centrifugation was the hypotonic solution replaced with a fixative (cooled methanol mixed with glacial acetic acid in a ratio of 3:1). The cells were centrifuged and the fixation process was repeated twice. A slightly turbid solution of suspended cells was prepared and two drops of fixed cells were spread onto clean microscope slides.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 well-spread metaphases containing 22 ± 2 centromeres were analysed per each concentration, solvent and positive controls equally divided among the duplicates (150 metaphases per group – replicate culture A and 150 metaphases per group – replicate B) by microscopic examination (1000x magnification).

DETERMINATION OF CYTOTOXICITY
- Method: other: relative increase in cell counts (RICC)
Evaluation criteria:
Positive response
The test item was considered to have caused a positive response in this assay if the test item showed a statistically significant increase (p < 0.05) over that of the solvent control group in the percentage of aberrant cells at one or more concentrations. The increase was concentration-related when evaluated with an appropriate trend test and any of results were outside distribution of the historical negative control data.

Negative response
The test item was considered to have caused a negative response if none of the test concentrations showed a statistically significant increase in the percentage of aberrant cells. There was no concentration-related increase when evaluated with an appropriate trend test and all results were inside the distribution of the historical negative control data.
Statistics:
The percentage of all aberrant cells as well as the total number of aberrations were taken as the statistical variable to analyze. Each treatment group (including the positive control group) was compared individually to the solvent control group. The data in each outcoming contingency table were evaluated statistically applying Chi-square test of independence. Significance level of 0.05 was adopted among statistical analyses to make appropriate conclusions. The test item treatment group was considered statistically different from the solvent one at P<0.05.
Possible dose-related trend was assessed applying regression analysis. Statgraphics™ Centurion, version XV software was applied for the purpose of statistical evaluation.

Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity was seen in all groups (3 h with and without metabolic activation, 21 h without metabolic activation)
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: No marked changes of pH of treatment media were observed in the experiments at concentrations up to 65.8 µg/mL tested as compared to the concurrent solvent controls.
- Effects of osmolality: The osmolality values were within the physiological ranges for these cells. The negative control cultures showed optimal growth under these conditions.
- Precipitation: No precipitate of test item was observed throughout the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC

HISTORICAL CONTROL DATA: The percentages of cells with aberrations were inside the historical negative control data.





Table 1. Mean values (% Cells with Aberrations)

3 Hours Treatment

Without Activation

With Activation

Sample (µg/mL)

Cells with Chromosome Aberrations (%)

Sample (µg/mL)

Cells with Chromosome Aberrations (%)

Solvent control

1.0

Solvent control

2.0

Test substance (1.18)

1.3

Test substance (18.81)

1.3

Test substance (2.35)

1.0

Test substance (21.15)

1.3

Test substance (3.76)

1.3

Test substance (23.5)

2.3

MMC (0.5)

20.3***

CP (40)

30.0 ***

MMC (mitomycin C), CP (cyclophosphamide)

*Statistically significant response using the Chi- square test (***p<0.001)

 

Table 2. Mean values (% Cells with Aberrations)

21 Hours Treatment

Without Activation

Sample (µg/mL)

Cells with Chromosome Aberrations (%)

Solvent control

1.7

Test substance (3.29)

1.3

Test substance (4.70)

1.7

Test substance (6.58)

0.7

MMC (0.07)

21.0***

MMC (mitomycin C)

*Statistically significant response using the Chi- square test (***p<0.001)

Conclusions:
Based on the results of the conducted study the test item did not exhibit clastogenic properties in mammalian cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jun - 06 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovak National Accreditation Service, Bratislava, Slovak Republic
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures), UK (originally established by Ford and Yerganian in 1958)
- Number of passages if applicable: 3 - 4
- Methods for maintenance in cell culture if applicable: Subculturing was performed 2-3 times a week with the aid of trypsin (0.25%)/EDTA solution and counted in suspension using Bürker’s chamber
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine, 100 units/mL penicillin adn 100 µg/mL streptomycin. 10% fetal bovine serum was added to growth media.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 20-methylcholanthrene
Test concentrations with justification for top dose:
Cytotoxicity range finder:
(-S9): 0, 0.094, 0.94, 9.4, 47, 94, 235, 470, 940 and 1880 µg/mL
(+S9): 0, 4.7, 9.4, 23.5, 47, 70.5, 94, 141, 235 and 470 µg/mL

Mutagenicity assays:
Experiment 1 (-S9): 0, 7.05, 14.1, 28.2 and 56.4 µg/mL
Experiment 2 (-S9): 0, 2.35, 4.7, 9.4, 18.8, 23.5; 28.2, 37.6 and 47 g/mL
Experiment 3 (+S9): 0, 11.75, 23.5, 47, 56.4, 75.2 and 84.6 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: ethanol with Tween 80 (10 mg/mL)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol with Tween 80 (10 mg/mL)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: at least 1 000 000 cells

DURATION
- Preincubation period: 24 h
- Exposure duration: 3 h with and without metabilic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency
Evaluation criteria:
The test item is considered to induce forward mutation at the hprt locus in Chinese hamster lung V79 cells if:
- At least one of the test concentrations exhibits a statistically significant increase in mutant frequency compared with concurrent negative control. The induced mutant frequency is at least 3-fold greater than that of the negative control.
- The increase is concentration-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
Statistics:
For each test concentration summary statistics (average, standard deviation, coefficient of variation, minimum, maximum) was calculated. The same was done for negative and positive control data. Two sample comparison was carried out to identify the test concentration for which median of the observed frequency data is significantly different from the median in the negative control group. Mann Whitney (Wilcoxon) W test was applied for this purpose. Significance level of 0.05 was adopted among statistical analyses to make appropriate conclusions.
Statgraphics TM Centurion software (version XV) was employed for statistical data evaluation
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes of pH of treatment media were observed in the mutation experiments at concentrations up to 94 µg/mL tested as compared to the concurrent vehicle controls.
- Effects of osmolality: The osmolality values were within the physiological ranges for these cells.
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: In the initial cytotoxicity range finding experiments up to nine concentrations were tested in the absence and presence of S9 ranging from 0.094 to 1880 μg/mL and 4.7 to 470 μg/mL respectively. At 94 µg/mL cloning efficacy was reduced to ~58% and ~11% in the absence and presence of S9, respectively. The acceptable reduction in relative survival (10-20%) was achieved in the case of the +S9 data. The cloning efficacies are shown in Table 4.

HISTORICAL CONTROL DATA: see Tables 1-3

ACCEPTANCE CRITERIA:
The assay is considered valid if the following criteria were met:
• Number of spontaneous mutants of the solvent control cultures are within the range of historical control data.
• The number of induced mutants of positive controls are within the expected range.
• A minimum of four analysable concentrations with mutant frequency data are required.

All acceptance criteria were fulfilled.

Table 4. % Relative plating efficacy values – Range Finding Experiment

Treatment

(µg/mL)

3 hour –S9

RF Exp 1b

3 hour +S9 RF Exp 2b

Vehiclea

100.0

100.0

TI (0.094)

117.6

-

TI (0.940)

113.9

-

TI (4.7)

-

121.1

TI (7.05)

-

-

TI (9.4)

85.5

116.4

TI (14.1)

-

-

TI (23.5)

-

101.2

TI (28.2)

-

-

TI (47.0)

37.1

66.5

TI (56.4)

-

-

TI (70.5)

-

64.8

TI (84.6)

-

-

TI (94.0)

58.2

10.6

TI (141.0)c

-

0

TI (235.0)c

0

0

TI (470.0)c

0

0

TI (940.0)c

0

-

TI (1880.0)c

0

-

a  Ethanol with Tween 80;

TI Test Item

b  5 plates counted/treatment level

c tested concentration was toxic after 3 hours treatment of V 79 cells

- not tested

Table 5. Summary of mutation data Experiment 1 (A, B)c without S9

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

 

 

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

Vehicleb

A

100.0

9.24

 

Vehicleb

B

100.0

10.13

TI (7.05)

A

92.8

7.47

 

TI (7.05)

B

80.6

12.46

TI (14.1)

A

70.1

10.75

 

TI (14.1)

B

72.6

11.30

TI (28.2)

A

45.4

8.77

 

TI (28.2)

B

33.9

12.23

TI (56.4)

A

70.1

11.15

 

TI (56.4)

B

59.3

10.56

EMS (600)

A

109.3

157.45*

 

EMS (600)

B

92.0

198.78*

a 6TG resistant mutants/106 viable cells 7 days after treatment; b Ethanol with Tween 80 ; TI -Test Item;

c  A or B - codes for two replicate cultures

*  Statistically significant (p<0.05) (Mann-Whitney W test)

Table 6. Summary of mutation data Experiment 2 (A, B)c without S9

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

 

 

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

Vehicleb

A

100.0

7.98

 

Vehicleb

B

100.0

9.19

TI (2.35)

A

100.0

8.56

 

TI (2.35)

B

95.5

12.38

TI (4.70)

A

95.9

4.53

 

TI (4.70)

B

92.0

5.66

TI (9.40)

A

74.8

6.29

 

TI (9.40)

B

89.4

8.30

TI (18.80)

A

45.0

5.48

 

TI (18.80)

B

46.2

7.08

TI (23.50)

A

51.9

3.89

 

TI (23.50)

B

57.7

7.02

TI (28.20)

A

44.1

8.55

 

TI (28.20)

B

55.1

6.35

TI (37.60)

A

35.6

4.21

 

TI (37.60)

B

41.6

3.59

TI (47.00)

A

20.1

3.49

 

TI (47.00)

B

19.2

5.14

EMS (600)

A

95.9

171.77*

 

EMS (600)

B

94.9

172.31*

a 6TG resistant mutants/106 viable cells 7 days after treatment;b Ethanol with Tween 80 ; TI -Test Item;

c  A or B - codes for two replicate cultures

*  Statistically significant (p<0.05) (Mann-Whitney W test)

Table 7. Summary of mutation data Experiment 3 (A, B)c with S9

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

 

 

Treatment
(µg/mL)

 

3 hour – S9
% Cloning eff.

MFa

Vehicleb

A

100.0

13.60

 

Vehicleb

B

100.0

13.77

TI (11.75)

A

113.8

19.44

 

TI (11.75)

B

96.8

10.68

TI (23.50)

A

111.7

37.89*

 

TI (23.50)

B

99.0

51.03*

TI (47.00)

A

45.0

11.99

 

TI (47.00)

B

29.3

9.43

TI (56.40)

A

28.4

9.24

 

TI (56.40)

B

27.4

20.69

TI (75.20)

A

20.2

8.72

 

TI (75.20)

B

13.9

10.24

TI (84.60)

A

17.3

13.77

 

TI (84.60)

B

9.8

4.14

DMBA (5)

A

77.9

137.93*

 

DMBA (5)

B

69.1

135.42*

a 6TG resistant mutants/106 viable cells 7 days after treatment;b Ethanol with Tween 80 ; TI -Test Item;

c  A or B - codes for two replicate cultures

*  Statistically significant (p<0.05) (Mann-Whitney W test)

Table 8. Average mutation frequencyc

Experiment 1
without S9

Experiment 2
without S9

Experiment 3
with S9

Treatment

(µg/mL)

MFa

Treatment

(µg/mL)

MFa

Treatment

(µg/mL)

MFa

Vehicleb

9.69

Vehicleb

8.59

Vehicleb

13.69

TI (7.05)

9.97

TI (2.35)

10.47

TI (11.75)

15.06

TI (14.1)

11.03

TI (4.70)

5.10

TI (23.50)

44.46*

TI (28.2)

10.5

TI (9.40)

7.30

TI (47.00)

10.71

TI (56.4)

10.86

TI (18.80)

6.28

TI (56.40)

14.97

 

-

TI (23.50)

5.46

TI (75.20)

9.48

-

-

TI (28.20)

7.45

TI (84.60)

8.96

-

-

TI (37.60)

3.90

-

-

-

-

TI (47.00)

4.32

-

-

EMS (600)

178.12*

EMS (600)

172.04*

DMBA (5)

136.68*

a 6TG resistant mutants/106viable cells 7 days after treatment;b Ethanol with Tween 80 ; TI -Test Item;

c   Data represented the average of determinations made in duplicate cultures

- not tested

*  Statistically significant (p<0.05) (Mann-Whitney W test)

Conclusions:
The test item was tested for its potential to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung cells according to OECD 476. Cells were treated with the test item with and without metabolic activation at concentrations of up to 84.6 µg/mL. Based on the results of the conducted study the test item did not exhibit mutagenic properties in mammalian cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria (Ames test)

The in vitro genetic toxicity of the test substance was assessed in a bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and in compliance with GLP (Hameln, 2018). A range-finding assay was first performed with S. typhimurium strains TA 97 and TA 100 using concentrations in the range of 50 - 5000 µg/plate without metabolic activation. As no inhibition of bacteria growth was observed, concentrations up to 5000 µg/plate (50, 150, 500, 1500 and 50000 µg/plate in DMSO) were tested in the main assay experiments using the Salmonella typhimurium strains TA 97, TA 98, TA 100, TA 1535 and E.coli WP2 uvr A. For the first main experiment the plate incorporation method was used with and withoud metabolic activation, the second main assay used the preincubation method without metabolic activation. The test substance did not exhibit mutagenic or cytotoxic properties in the absence or presence of metabolic activation. The vehicle and positive controls were valid and lay within the range of historical control data, proving the validity of the assay. Based on the results of the conducted study, the test substance is not considered to exhibit mutagenic properties in bacterial cells.

Clastogenic potential in mammalian cells

The in vitro clastogenic toxicity of the test substance was assessed in a mammalian chromosome aberration test in Chinese hamster lung (V79) cells according to GLP criteria and OECD guideline 473 (Hameln, 2018). The test item was formulated in ethanol in the presence of Tween 80. Cells were incubated for 3 hours with the test substance at concentrations of 1.18, 2.35, 3.76, 4.23 and 4.70 µg/mL without metabolic activation and at concentrations of 9.40, 11.28, 13.16, 14.10, 15.04, 16.92, 17.86, 18.80, 21.15, 23.50, 25.85 and 28.20 µg/mL with metabolic activation. Cells were harvested after an 18-hour recovery period. Cells were also exposed for 21 hours (1.5 length cell cycle) at concentrations of 0.94, 3.29, 4.70, 6.11, 6.58, 7.05 and 7.52 µg/mL without metabolic activation. Toxicity was determined by Relative Increase in Cell Counts (RICC) in the test item treated cells, as compared to the cells treated with the solvent control. Significant cytotoxicity was observed in all evaluated experiments, 3-hour both with and without metabolic activation and also at the extended 21- hour treatment. Mitomycin C (MMC) and cyclophosphamide (CP) were used as the positive controls in the non- activated and activated systems, respectively. Chromosome aberrations were scored from the cells treated for 3 hours with the concentrations of 1.18, 2.35 and 3.76 µg/mL without activation and with the concentrations of 18.8, 21.15 and 23.5 µg/mL with activation. In the experiment with extended treatment, chromosome aberrations were scored for the concentrations of 3.29, 4.70 and 6.58 µg/mL. The corresponding solvent control and one concentration each of the positive controls (MMC at 0.5 µg/mL for 3-hour treatment, at 0.07 µg/mL for 21-hour treatment and CP at 40 µg/mL) were also scored. Three hundred metaphases were scored from each test item concentration and the controls. The results from the 3-hour treatments with and without metabolic activation as well as the extended treatment indicate that the tested concentrations, did not induce an increase in the percentage of cells with aberrations compared to the solvent controls. Results of positive and solvent controls confirmed the validity of the assay. Based on these results, the test substance has no clastogenic potential in this mammalian chromosome aberration test.

Gene mutation in mammalian cells

The in vitro potential the test substance to induce reverse mutations at the HPRT locus in V79 Chinese hamster lung cells was assessed according to GLP criteria and OECD guideline 476 (Hameln, 2018). The test item was formulated in ethanol in the presence of Tween 80. In a preliminary cytotoxicity test, treatment concentrations of the test item of up to 1880 µg/mL in the presence and absence of S9 were applied followed by a 3 h incubation. After treatment the test item was removed by rinsing with Dulbecco's Phosphate Buffered Saline. Subsequently, the cells were trypsinized, suspended in complete culture medium and incubated for 7 days. After staining with 1% methylene blue, viable colonies were identified visually and counted. Osmolality and pH in the test medium did not change at any concentration and no precipitation was noted. Relative survival (RS) was reduced to ~58% and ~11% at a test item concentration of 94 µg/mL in the absence and presence of S9, respectively.

Accordingly, for Experiment 1 with duplicate cultures, seven concentrations of test item ranging from 7.05 to 112.8 µg/mL were tested in the absence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the absence of S9 was 56.4 µg/mL, which gave RS values of ~70% and ~59%, respectively. As an inadequate level of cytotoxicity was obtained, a second experiment in the absence of S9 was performed. In Experiment 2 with duplicate cultures, fourteen concentrations of test item ranging from 0.94 to 94 µg/mL were tested in the absence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the absence of S9 was  47 µg/mL, which gave RS values of ~20% and ~19%, respectively.

 In Experiment 3 with duplicate cultures, seven concentrations ranging from 11.75 to 94 µg/mL were tested in the presence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the presence of S9 was 84.6 µg/mL, which gave RS values of ~17% and ~10%, respectively.

In Experiments 1 and 2 with duplicate cultures in the absence of S9, no statistically significant increases in mutant frequencies were observed following treatment with the test item at any concentration tested. In Experiment 3 with duplicate cultures in the presence of S9, statistically significant increases (p< 0.05) in mutant frequencies were observed following treatment at a concentration of 23.5 µg/mL only. At this level RS was not reduced (~112 and ~99%). The observed increase in mean mutant frequency was 3.2-fold above that of the concurrent vehicle control, with no evidence of an increase in mutant frequencies at any of the other concentrations tested.

Vehicle and positive control treatments were included in each mutation experiment in the absence and presence of S9. Mutant frequencies in vehicle control were consistent with the acceptable range and clear increases in mutant frequency were induced by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a)anthracene (with S9). The assay system was therefore considered to be both sensitive and valid.

Under the conditions of this test, the test item was evaluated as not mutagenic in mammalian cells.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.