Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

Under the conditions of the test, the test material was found to be negative for mutagenicity in both the S. typhimurium and E. coli strains tested in the presence and absence of metabolic activation.

Mouse lymphoma assay

Under the experimental conditions of this study, the test material did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolising system.

Micronucleus test in cultured human lymphocytes

Under the experimental conditions of the study, the test material, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using human lymphocytes, either in the presence or absence of a rat liver metabolising system.

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:
21 December 1998 to 21 January 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted in 1998 to 1999 in accordance with OECD Guideline 471/472 on the appropriate strains of bacteria (S. typhimuriumTA1535, TA100, TA1537 and TA98 as well as E. coli WP2uvrA in both the presence and absence of metabolic activation. Whilst not conducted to GLP the report does include a signature page confirming that the report accurately reflects the proceedings of the experiment signed by the laboratory manager. Neither the batch details nor the purity of the substance are reported. Concurrent controls were run as a part of the experiment. Six dose levels were tested during the study, however the experiments were run on duplicate rather than triplicate plates and whilst the study result in an unequivocally negative result, the experiment was not duplicated and no justification for this was given in the report. Despite its basic nature and some deviations from the approved guidelines the study can be considered to be reliable with restrictions.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Plates were tested in duplicate not triplicate and the negative result was not confirmed
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
yes
Remarks:
Plates were tested in duplicate not triplicate and the negative result was not confirmed
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine synthesis in S. typhimurium strains, and tryptophan synthesis in the E. coli strain tested.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Broth No. 2 (Oxoid)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient Broth No. 2 (Oxoid)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Range finding test: 0, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Main test: 0, 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: Performed in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: assessment of the bacterial lawn growth
Evaluation criteria:
Plates were assessed for a significant increase in the number of revertant colonies.
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:
Observed at the two highest doses tested
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:
Observed at the two highest doses tested
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Based on the results of the range finding test, the maximum concentration selected for the main test was 1250 µg/plate as the test material caused a reduction in the growth of the bacterial lawn at concentrations above this dose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Dose response curves for the number of revertants both with and without metabolic activation are presented in the attached figures.

Table 1: Results of the main test

Metabolic activation

Test material concentration

Replicate

Number of revertant colonies per plate

Base-pair substitution

Frameshift

TA 100

TA 1535

WP-2uvrA

TA 98

TA 1537

- S9 Mix

Solvent control

R1

148

9

17

16

9

R2

156

7

17

12

14

Mean

(152)

(8)

(17)

(14)

(12)

39.1

R1

138

11

14

14

13

R2

160

13

15

17

6

Mean

(149)

(12)

(15)

(16)

(10)

78.1

R1

159

6

14

17

13

R2

155

11

18

15

11

Mean

(157)

(9)

(16)

(16)

(12)

156

R1

152

6

16

22

6

R2

152

8

22

14

9

Mean

(152)

(7)

(19)

(18)

(8)

313

R1

151

9

17

7

7

R2

166

8

19

7

8

Mean

(159)

(9)

(18)

(7)

(8)

625

R1

62*

6*

13*

0*

2*

R2

82*

4*

10*

5*

2*

Mean

(72)

(5)

(12)

(3)

(2)

1250

R1

0*

0*

0*

0*

0*

R2

0*

0*

0*

0*

0*

Mean

(0)

(0)

(0)

(0)

(0)

+ S9 Mix

Solvent control

R1

173

9

19

24

12

R2

165

9

19

23

11

Mean

(169)

(9)

(19)

(24)

(12)

39.1

R1

152

8

16

31

11

R2

167

8

1

28

11

Mean

(160)

(8)

(16)

(30)

(11)

78.1

R1

158

13

30

33

9

R2

198

16

11

26

13

Mean

(178)

(15)

(21)

(30)

(11)

156

R1

152

12

23

31

19

R2

164

16

26

30

9

Mean

(158)

(14)

(25)

(31)

(14)

313

R1

170

3

14

19

16

R2

163

8

11

28

15

Mean

(167)

(6)

(13)

(24)

(16)

625

R1

150*

7*

18*

19*

9*

R2

128

5*

23*

18*

4*

Mean

(117)

(6)

(21)

(19)

(7)

1250

R1

122*

7*

12*

15*

3*

R2

125

4*

8*

20*

1*

Mean

(124)

(6)

(10)

(18)

(2)

Positive controls

- S9 Mix

 

 

 

Positive control substance

AF-2

NaN3

AF-2

AF-2

9-AA

Concentration (µg/plate)

0.01

0.5

0.01

0.1

80

 

R1

486

488

93

264

259

R2

516

491

85

263

285

Mean

(501)

(490)

(89)

(264)

(272)

Positive controls

+ S9 Mix

 

 

 

Positive control substance

2-AA

2-AA

2-AA

2-AA

2-AA

Concentration (µg/plate)

1.0

2.0

10

0.5

2.0

 

R1

826

286

1292

239

116

R2

867

311

1369

255

112

Mean

(847)

(299)

(1331)

(247)

(114)

AF-2 = 2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide

NaN3= sodium azide

9-AA = 9-aminoacridine

2-AA = 2 -aminoanthracene

*Inhibition of bacterial growth

Conclusions:
Interpretation of results:
negative Both with and without metabolic activation

Under the conditions of the test, the test material was found to be negative for mutagenicity in both the S. typhimurium and E. coli strains tested in the presence and absence of metabolic activation.
Executive summary:

The mutagenicity of the test material was assessed using a bacterial reverse mutagenicity assay (Ames test). The study was performed in compliance with the current OECD guidelines 471 and 472 with a few minor deviations from the standard protocol. Under the conditions of the test, the test material was found to be negative in all strains tested both with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 February 2017 - 23 May 2017
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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose level for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).
The selected dose levels were ranged from 10 to 400 µg/mL.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice according to available solubility data. Test item was soluble in the vehicle at the concentration of 200 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethane sulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
- Any supplementary information relevant to cytotoxicity:
Evaluation criteria:
IWGT recommendations (d, e, f) were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20 %, are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20 % if:
- there is at least one negative data point between 20 and 25 % Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20 % Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25 % and there is also a negative data point between 10 and 1% Adj. RTG.
Statistics:
yes: to assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG ≥ 10 %.
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: An emulsion was noted in the culture medium at the end of the treatment period, at dose levels ≥ 270 µg/mL.

RANGE-FINDING STUDIES: Using a test item concentration of 200 mg/mL and a treatment volume of 1% (v/v) in the culture medium (i.e. 200 µL/20 mL culture medium) the highest recommended dose level of 2000 µg/mL was achievable. Thus the dose levels selected for the treatments of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest tested dose level, i.e. 2000 µg/mL, the pH of the culture medium was approximately 7.9 (7.6 for the vehicle control) and the osmolality was 425 mOsm/kg H2O (451 mOsm/kg H2O for the vehicle control). Thus none of the selected dose levels was considered to produce extreme culture conditions.

At the end of the treatment period, an emulsion was observed in the culture medium at dose levels ≥ 400 µg/mL.

Following the 3-hour treatments without and with S9 mix, a severe cytotoxicity was observed at dose levels ≥ 400 µg/mL, as shown by a 100% decrease in the Adj. RTG.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see document attached
Conclusions:
The test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.

 

Methods

After a preliminary cytotoxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105 cells/mL were exposed for 3 hours to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2 humidified incubator.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Results

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

 

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose level for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

 

Experiment without S9 mix

The selected dose levels were 10, 20, 40, 80, 160, 240, 320 and 400 µg/mL.

 

An emulsion was noted in the culture medium at the end of the treatment period at dose levels ≥ 320 µg/mL. This emulsion did not prevent any scoring.

 

Cytotoxicity

A moderate to severe cytotoxicity was induced at dose levels ≥ 160 µg/mL, as shown by a 50 to 100% decrease in the Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response.

Experiments with S9 mix

In first instance, the selected dose levels were 10, 20, 40, 80, 160, 240, 320 and 400 µg/mL. Then, since the highest analyzable dose level did not show precipitation or emulsion in the culture medium and did not induce the recommended level of cytotoxicity (i.e. an Adj. RTG value between 10 and 20%), a second experiment was undertaken using a modified range of dose levels as follows:10, 20, 50, 100, 250, 270, 300 and 400 µg/mL.

 

An emulsion was noted in the culture medium at the end of the treatment period, at dose levels ≥ 270 µg/mL. This emulsion did not prevent any scoring.

 

Cytotoxicity

In the first experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 240 µg/mL, as shown by a 60 to 100% decrease in the Adj. RTG.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 250 µg/mL, as shown by a 47 to 100% decrease in the Adj. RTG.

 

Mutagenicity

In both experiments, no noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle controls, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response.

Experiments with S9 mix

In first instance, the selected dose levels were 10, 20, 40, 80, 160, 240, 320 and 400 µg/mL. Then, since the highest analyzable dose level did not show precipitation or emulsion in the culture medium and did not induce the recommended level of cytotoxicity (i.e. an Adj. RTG value between 10 and 20%), a second experiment was undertaken using a modified range of dose levels as follows:10, 20, 50, 100, 250, 270, 300 and 400 µg/mL.

 

An emulsion was noted in the culture medium at the end of the treatment period, at dose levels ≥ 270 µg/mL. This emulsion did not prevent any scoring.

 

Cytotoxicity

In the first experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 240 µg/mL, as shown by a 60 to 100% decrease in the Adj. RTG.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 250 µg/mL, as shown by a 47 to 100% decrease in the Adj. RTG.

Mutagenicity

In both experiments, no noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle controls, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 February 2017 - 07 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human lymphocytes (primary cell cultures)
Details on mammalian cell type (if applicable):
Obtained from young (i.e. 18 to 35 years old), healthy, non-smoking donors and collected into heparinized sterile tubes.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasine B (CytoB)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The test item was diluted in the vehicle at concentrations of:
 200 mg/mL for the preliminary cytotoxicity test,
 100 mg/mL for the first experiment,
 75 mg/mL for the second experiment.
top dose: highest analyzable dose level and cytotoxic dose levels.
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchichine
Details on test system and experimental conditions:
The test item was diluted in the vehicle at concentrations of:
 200 mg/mL for the preliminary cytotoxicity test,
 100 mg/mL for the first experiment,
 75 mg/mL for the second experiment.
Rationale for test conditions:
Cytotoxicity (or cytostasis) was shown by the decrease in the RI when compared to the vehicle control culture.
Each main experiment was considered valid if the following criteria were met:
-the mean frequency of cells that have undergone mitosis (binucleated + multinucleated cells) in the vehicle control cultures is at least 50%,
-the mean background frequency of Micronucleated Binucleated Cells in the vehicle control cultures should be consistent with the historical vehicle control range for the Laboratory,
-a statistically significant increase in the frequency of MNBC has to be obtained in the positive controls over the background frequency of the vehicle control cultures.
Evaluation criteria:
The biological relevance of the results was considered first.
Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic potential if, in any of the experimental conditions examined, all the following criteria are met:
-a statistically significant increase in the frequency of MNBC, in comparison to the corresponding vehicle control, is obtained at one or more dose levels,
-a dose-response relationship (dose-related increase in the frequency of MNBC) is demonstrated by a statistically significant trend test,
-for at least one dose level, the frequency of MNBC of each replicate culture is above the corresponding vehicle historical range.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response are met.
When the criteria of a positive response were only partially met, results were evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments.
Statistics:
Treated cell cultures were compared to that of the vehicle control cell cultures. Unless treated culture data were lower than or equal to the vehicle control data, the statistical comparison was performed using the chi2 test, in which p = 0.05 was used as the lowest level of significance.
To assess the dose-response trend, a linear regression was performed between the mean frequencies of Micronucleated Binucleated Cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.
Key result
Species / strain:
lymphocytes: human lymphocytes (primary cell cultures)
Metabolic activation:
with and without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
See the result tables in the attachement.

Experiments without S9 mix: Neither statistically significant nor dose-related increase in the frequency of MNBC was noted at any of the analyzed dose levels relative to the vehicle control (p>0.05). Moreover, none of the analyzed dose levels showed frequency of MNBC of both replicate cultures above the vehicle reference data range. These results met the criteria for a negative response.

Experiments with S9 mix: Neither statistically significant nor dose-related increase in the frequency of MNBC was noted at any of the analyzed dose levels relative to the vehicle control (p>0.05). Moreover, none of the analyzed dose levels showed frequency of MNBC of both replicate cultures above the vehicle reference data range. These results met the criteria for a negative response.
None of the analyzed dose levels induced the recommended level of cytotoxicity (i.e. 55 ± 5%), however, considering the lack of BC at the dose level of 437.5 µg/mL, as well as the narrow dose levels spacing used in the second experiment, available results were considered as suitable to allow a reliable interpretation.

PRELIMINARY CYTOTOXICITY TEST:

dose levels: 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest tested dose level,i.e.2000 µg/mL, the pH of the culture medium was approximately 7.7 (as for the vehicle control) and the osmolality was 423mOsm/kg H2O (455mOsm/kg H2O for the vehicle control).Therefore, none of the selected dose levels was considered to produce extreme culture conditions.

An emulsion was observed in the culture medium at dose levels ≥

1000 µg/mL at the end of the 3‑hour treatment period and at 2000 µg/mL at the end of the 24-hour treatment period.

Following the 3-hour treatment with and without S9 mix, a severe cytotoxicity was observed at dose levels≥ 1000 µg/mL, as shown by a 100% decrease in the RI.

Following the 24-hour treatment (without S9 mix), a severe cytotoxicity was observed at dose levels≥ 200 µg/mL, as shown by a 91 to 100% decrease in the RI.

MAIN CYTOGENETIC EXPERIMENTS:

dose levels:

.         31.3, 62.5, 125, 250, 500, 750 and 1000 µg/mL for the 3-hour treatments with and without S9 mix (first experiment)

.         3.91, 7.81, 15.6, 31.3, 62.5, 125 and 250 µg/mL for the continuous 24-hour treatment (without S9 mix) ( first experiment)

.         62.5, 125, 250, 312.5, 375, 437.5, 500 and 750 µg/mL for the 3-hour treatments with and without S9 mix (second experiment)

.         6.25, 12.5, 25, 37.5, 50, 62.5, 75 and 125 µg/mL for the continuous 24-hour treatment (without S9 mix) (second experiment)

Experiments without S9 mix

 Cytotoxicity

Following the 3-hour treatment, a moderate to severe cytotoxicity was observed at dose levels≥ 437.5 µg/mL, as shown by a 50 to 100% decrease in the RI,

Following the 24-hour treatment, a moderate to severe cytotoxicity was observed at dose levels≥ 50 µg/mL, as shown by a 42 to 100% decrease in the RI.

 Micronucleus analysis

The dose levels selected for the micronucleus analysis were as follows:

.            62.5, 125 and 250 µg/mL for the 3-hour treatment in the first experiment, higher dose levels being too cytotoxic,

.            250, 312.5, 375 and 437.5 µg/mL for the 3-hour treatment in the second experiment, the latter inducing a 50% decrease in the RI relative to the vehicle control,

.            7.81, 15.6 and 31.3 µg/mL for the 24-hour treatment in the first experiment, higher dose levels being too cytotoxic,

.            25, 50 and 62.5 µg/mL for the 24-hour treatment in the second experiment, the latter inducing a 54% decrease in the RI relative to the vehicle control.

Experiments with S9 mix

Cytotoxicity

In both experiments, aseverecytotoxicity was observed at dose levels ≥500 µg/mL, as shown by a 100% decrease in the RI.

Micronucleus analysis

The dose levels selected for the micronucleus analysis were:

.             62.5, 125 and 250 µg/mL in the first experiment, higher dose levels being too cytotoxic,

.            250, 312.5, 375 and 437.5 µg/mL in the second experiment, the latter inducing a 31% decrease in the RI and higher dose levels being too cytotoxic.

Conclusions:
Under the experimental conditions of the study, the test item, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using human lymphocytes, either in the presence or absence of a rat liver metabolizing system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

IN VITRO GENE MUTATION STUDY IN BACTERIA

The key study Sasaki & Shirai (2000), provided to fulfil this endpoint was conducted in accordance with OECD 471/472, although no information relating to the GLP status was provided in the reporting of the study, quality assurances were reported by the laboratory. In line with the criteria for assessing data quality as described in Klimisch (1997), the study was assigned a reliability score of 2 as some deficiencies were present in the methodology, and considered reliable and adequate for assessment. This was selected as the key study for this endpoint as it was conducted on the neat substance and the test sample used is more representative of the registered substance. Under the conditions of the test, the test material was found to be negative for mutagenicity in both the S. typhimurium and E. coli strains tested in the presence and absence of metabolic activation.

 

Supporting information was available in the form of Watanabe & Moritomo (1989). The study was conducted to a method broadly in line with the methodologies presented in the standardised OECD Guideline 471. As with the key study, no information relating to the GLP status was provided in the reporting of the study, quality assurances were again reported by the laboratory. The study was therefore assigned a reliability score of 2 in accordance with the criteria for assessing data quality of Klimisch (1997). This study was provided as supporting information, as it was performed on a formulation of the registered substance and conclusions on the mutagenicity of the registered substance cannot be based on this study alone. The results of the study confirmed the findings of the key study. The test material was non-mutagenic under the conditions of the test.

Justification for selection of genetic toxicity endpoint

In line with the criteria for assessing data quality as described in Klimisch (1997), the study was assigned a reliability score of 2 (as some deficiencies were present in the methodology) and considered reliable and adequate for assessment. This was selected as the key study for this endpoint as it was conducted on the neat substance and the test sample used is more representative of the registered substance.

Short description of key information:

IN VITRO GENE MUTATION STUDY IN BACTERIA

Key study:- Sasaki & Shirai (2000), Bacterial Reverse Mutation Assay (Ames Test) (OECD 471/472): Negative (non-mutagenic)

Endpoint Conclusion: No adverse effect observed (negative)

MOUSE LYMPHOMA ASSAY

The objective of this study was to evaluate the potential of the test item, to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.

 

Methods

After a preliminary cytotoxicity test, the test item, diluted in dimethylsulfoxide (DMSO), was tested in two independent experiments with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105 cells/mL were exposed for 3 hours to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a 37°C, 5% CO2 humidified incubator.

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Results

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid.

Since the test item was found cytotoxic in the preliminary test, the selection of the highest dose level for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (decrease in the Adj. RTG).

 

Experiment without S9 mix

The selected dose levels were 10, 20, 40, 80, 160, 240, 320 and 400 µg/mL.

An emulsion was noted in the culture medium at the end of the treatment period at dose levels ≥ 320 µg/mL. This emulsion did not prevent any scoring.

 

Cytotoxicity

A moderate to severe cytotoxicity was induced at dose levels ≥ 160 µg/mL, as shown by a 50 to 100% decrease in the Adj. RTG.

 

Mutagenicity

No noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response.

 

Experiments with S9 mix

In first instance, the selected dose levels were 10, 20, 40, 80, 160, 240, 320 and 400 µg/mL. Then, since the highest analyzable dose level did not show precipitation or emulsion in the culture medium and did not induce the recommended level of cytotoxicity (i.e. an Adj. RTG value between 10 and 20%), a second experiment was undertaken using a modified range of dose levels as follows:10, 20, 50, 100, 250, 270, 300 and 400 µg/mL.

An emulsion was noted in the culture medium at the end of the treatment period, at dose levels ≥ 270 µg/mL. This emulsion did not prevent any scoring.

 

Cytotoxicity

In the first experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 240 µg/mL, as shown by a 60 to 100% decrease in the Adj. RTG.

In the second experiment, a moderate to severe cytotoxicity was induced at dose levels ≥ 250 µg/mL, as shown by a 47 to 100% decrease in the Adj. RTG.

 

Mutagenicity

In both experiments, no noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle controls, and no dose-response relationship was demonstrated by the linear regression. These results did not meet the criteria for a positive response.

 

Conclusion

Under the experimental conditions of this study, the test material did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolising system.

MICRONUCLEUS TEST IN CULTURED HUMAN LYMPHOCYTES

Under the experimental conditions of the study, the test material, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using human lymphocytes, either in the presence or absence of a rat liver metabolising system.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.