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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 27, 2014 to March 20, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Range finding test in strain TA100 and E.Coli WP2 uvrA with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330; 5000 ug/L

Experiment 1: Tester trains TA1535, TA1537, TA98 with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L.

Experiment 2: Tester trains TA1535, TA1537, TA98, TA100: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L. Tester strain E.Coli WP2 uvrA: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330 and 5000 ug/L
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535; concentration/plate 5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191
Remarks:
TA1537; concentration/plate 2.5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98; concentration/plate 10µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
TA100; concentration/plate 650µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA; concentration/plate 15µg
Positive controls:
yes
Remarks:
with metabolic activation and solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535 2.5µg S9-mix 5 and 10%; TA1537 2.5µg S9-mix 5%; TA1537 5µg S9-mix 10%; TA98 1µg S9-mix 5 and10%; TA100 1µg S9-mix 5% ; TA100 2µg S9-mix 10% ; W2P uvrA 15µg S9-mix 5 and 10%
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a)The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain

b)The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean c)The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extendto 5 mg/plate.

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is not greater than three (3) times the concurrent vehicle control.

b)The negative response should be reproducible in at least one independently repeated experiment.


A test substance is considered positive (mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent vehicle control.

b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.


Key result
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid

Dose range finding\Experiment 1

The test substance was tested in the tester strains TA100 and WP2 uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay at a concentration range of 1 to 333 µg/plate in the absence of S9-mix and at a concentration range of 3 to 1000 µg/plate in the presence of 5% (v/v) S9-mix with the tester strains, TA1535, TA1537 and TA98. The results are shown in Table 3 and Table 4 of the study report.  

 

Precipitate

Dose range finding test: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate.

 

First mutation experiment: No precipitation on the plates was observed at the start or at the end of the incubation period.

 

Toxicity

To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

 

In tester strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed. However, the test substance precipitated heavily on the plates at the test substance concentration of 3330 and 5000 μg/plate in the absence of S9-mix, therefore the number of revertant colonies could not be determined of these dose levels.

 

In tester strain TA98, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix.

 

The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains in the dose range finding/first mutation experiment is presented in Table1.

Table1: Toxicity of Amides, C8-18(even-numbered) and C18(unsatd.), N-(2-hydroxypropyl) in the dose range finding/first experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA100

100            slight                  -1

333-3330    extreme        microcolonies

5000          extreme                -2

 333          slight                   -1

1000-3330  extreme        microcolonies

5000          extreme               -2

TA1535

333             extreme        microcolonies

1000          moderate      microcolonies

TA1537

333            moderate      microcolonies

1000          extreme         microcolonies

-1  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

-2  Due to the amount of precipitate no colony determination was possible

Experiment 2

To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the following dose range was selected for the second mutation assay:

TA1535, TA1537, TA100: Without S9-mix: 1, 3, 10, 33, 100 and 333 μg/plate and with S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate

TA98 and WP2 uvrA: Without and with S9-mix: 33, 100, 333, 1000 and 3330 µg/plate

The results are shown in Table 5 of the study report.

Precipitate

Precipitation of the test substance on the plates was observed at the start of the incubation period at the concentration of 3330 µg/plate. At the end of the incubation period, precipitation on the plates was observed at concentrations of 1000 and 3330 µg/plate in the absence of S9-mix and at 3330 µg/plate in the presence of S9-mix.

 

Toxicity

In tester strain WP2 uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

 

The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains is presented in Table 2.

Table 2: The reduction of the bacterial background lawn and the reduction in the number of revertants in the second mutation experiment

 

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

333            moderate           -1

1000          moderate            -1

TA1537

333            moderate           extreme

1000          extreme        microcolonies

TA98

333            slight                 -1

1000          moderate           extreme

3330          extreme             complete

3330          moderate            extreme

TA100

100            slight                 moderate

333            moderate           extreme

1000          extreme        microcolonies

-1  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

Experiment 3

In the first experiment in tester strain TA98 no toxicity or precipitate on the plates was observed in the absence and presence of S9-mix. Therefore a third mutation experiment was performed with this strain at a concentration range of 333 to 3330 µg/plate. The results are shown in Table 6 of the study report.

Precipitate

Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate. In the absence of S9-mix, precipitation on the plates was also observed at the end of the incubation period at the concentration of 1000 µg/plate.

 

Toxicity

In the absence of S9-mix, a slight to extreme reduction of the bacterial background lawn was observed at test substance concentrations of 333 µg/plate and above. An extreme reduction in the number of revertants or no revertant colonies was observed at 1000 and 3330 µg/plate, respectively.

 

In the presence of S9-mix, a slight to extreme reduction of the bacterial background lawn was observed at test substance concentrations of 1000 and 3330 µg/plate. No revertant colonies were observed at the test substance concentration of 3330 µg/plate.

Conclusions:
Under the study conditions, the test substance is not considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance in a reverse mutation (Ames) assay with the tester strains Salmonella typhimurium TA 1535, TA 1537, TA98 and TA100 and Escherichia coli WP2 uvrA according to OECD Guideline 471.

In the dose range finding assay, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards in the presence of S9-mix. In strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5000 µg/plate, the number of revertant colonies of this dose level could not be determined. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 µg/plate in the presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains, except in tester strain TA98 in the presence of S9-mix.

In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 µg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 10 to 3330 µg/plate in strain WP2 uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitate on the plates was only observed at the dose level of 3330 μg/plate in the absence of S9-mix. Toxicity was observed in all strains, except in TA1535 and TA1537 in the presence of S9-mix and in WP2 uvrA in the absence and presence of S9-mix. 

Since in the first experiment in strain TA98 and in the second experiment in strains TA1537 and WP2 uvrA no toxicity or precipitate on the plates was observed in the presence of S9-mix, a third mutation experiment was performed with these and strain TA98 in the presence of S9-mix. The substance was tested up to 5000 µg/plate. There was precipitation on the plates at dose levels of 3330 and 5000 μg/plate, so that the bacterial background could not be determined at 3330 and 5000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in strain TA98 in the presence of 5 and 10 %(v/v) S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance is not considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay ( Verspeek-Rip, 2014).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From Aug. 15, 1997 to Sep. 1, 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only 4 strains tested
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene (his- to his+)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: TA 1537: his C 3076, rfa-, uvrB-; TA 98: his D 3052, rfa-, uvrB-, R-factor; TA 1535: his G 46; rfa-, uvrB-; TA 100: his G 46, rfa-, uvrB-, R-factor
Metabolic activation:
with and without
Metabolic activation system:
15% v/v rat liver S9 fraction in standard co-factors
Test concentrations with justification for top dose:
Preliminary test (Range-finding test): 3, 10, 33, 100, 333, 1,000, 2,500 and 5,000 μg/plate both with and without metabolic activation (only TA 98 and TA 100 tested)

Main mutation test: 33, 100, 333, 1,000, 2,500 and 5,000 μg/plate both with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties. No visible precipitation of the test material occurred up to the highest investigated dose.
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: 10 μg/plate for TA 1535 and TA 100
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine; 10 μg/plate for TA 98 and 50 μg/plate for TA 1537
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; 2.5 μg/plate for TA 1535, TA 1537, TA 98 and TA 100
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation) for preliminary test; in agar (plate incorporation) and pre-incubation for main test

DURATION
- Pre-incubation period: 60 min at 37 °C (applicable only for pre-incubation test)
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneous revertants and clearing of the background lawn were taken as measure of cytotoxicity.





Evaluation criteria:
A test article is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or biologically relevant and reproducible increase for at least one concentration is induced.

A response is called biologically relevant if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice in TA 1535 and TA 1537.

A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.
Statistics:
None
Key result
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:
The background growth was reduced from 1,000 up to 5,000 μg/plate with and without metabolic activation in all the strains used; see Table 4 for concentrations causing reduction in spontaneous revertants
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The dose range for the main test was determined in a preliminary toxicity test using TA 98 and TA 100 strains only. The results are as follows:
- The test material caused reduction in the number of revertants from 1,000 up to 5,000 μg/plate without metabolic activation in TA 98 stain only.
- The background growth was reduced from 1,000 up to 5,000 μg/plate with and without metabolic activation in both the strains used.  
- No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
- The dose range selected for the main study was 33-5,000 μg/plate.






Table 2. Mean revertants per plate in plate incorporation test

Substance

Revertants per plate

(mean ± sd)

TA 1535

TA 1537

TA 98

TA 100

-

+

-

+

-

+

-

+

Negative control

14±2.6

18±1.5

7±2

13±2.1

25±4.5

33±6.8

112±14.5

134±14.4

Solvent control

13±1.5

16±1

6±1.2

12±2

27±2

25±3.1

116±5.2

161±4.2

Positive control

1037±50.6

195±11.5

138±13.1

80±8

566±18.5

864±13.1

980±61

1261±227.4

Test substance

(µg/plate)

33

13±2

15±3.5

7±2.1

10±3.6

22±4.6

25±6.8

134±9.7

139±22.9

100

14±3

16±2.5

7±1.5

11±1.5

22±2.3

26±3.5

127±14.6

130±6.7

333

10±2.1

16±3.1

6±2.1

12±2.5

20±1.5

28±3

122±10.3

123±5.8

1,000

12±2.1

13±3.2

6±0.6

10±2.1

14±6.7

29±7.6

131±11.9

135±10.2

2,500

7±1

6±1.7

4±1

8±1.5

5±2.5

15±2.6

107±12

118±15.4

5,000

2±1

4±2.1

2±1.2

4±2

4±3.6

16±6.2

100±17.1

157±16.6

Table 2. Mean revertants per plate in pre-incubation test

Substance

Revertants per plate

(mean ± sd)

TA 1535

TA 1537

TA 98

TA 100

-

+

-

+

-

+

-

+

Negative control

22±3.2

25±9.5

11±3.6

20±1.5

24±6.7

34±5.9

114±13.2

152±3.8

Solvent control

23±7.5

29±12.5

15±3.5

22±10

19±6

38±6.8

115±4.2

153±15.9

Positive control

1127±18.4

179±12

123±9.3

84±5

552±30.3

1098±269.4

973±153.2

647±58.4

Test substance

(µg/plate)

33

22±2.9

24±3.5

11±4.2

20±1.5

20±6

31±3.1

106±11

151±3.5

100

23±4.5

18±5.2

8±2.9

22±7.8

20±2.5

34±5

111±10.6

154±8.2

333

16±1.2

19±9.5

4±3.8

21±2.6

18±4.6

33±1.2

101±15

160±8.2

1,000

20±3.1

11±4

7±1

20±8.1

24±4.7

28±1.5

87±14.5

135±11.6

2,500

9±4

8±2.1

4±1.5

22±6.4

16±2.1

27±9.7

56±48.8

135±13.5

5,000

7±1

11±1.5

2±1.2

16±3.6

7±1.5

13±2.1

78±13.1

95±5.9

Table 3. Concentrations causing reduction in the number of spontaneous revertants

Strain

Toxic (reduction in no. of spontaneous revertant) concentration (µg/plate)

Plate incorporation test

Pre-incubation test

with S9 mix (+)

without S9 mix (-)

with S9 mix (+)

without S9 mix (-)

TA 1535

2,500 – 5,000

2,500 – 5,000

1,000 – 5,000

2,500 – 5,000

TA 1537

5,000

5,000

-

100 – 5,000

TA 98

-

1,000 – 5,000

5,000

5,000

TA 100

-

-

-

2,500

Conclusions:
Under the conditions of the study, the test substance was considered to be non-mutagenic.

Executive summary:

An OECD Guideline 471 study was conducted to determine the mutagenic potential of the test substance using a bacterial/microsome test system. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to the substance using both the plate incorporation method and pre-incubation method at six concentrations (33, 100, 333, 1,000, 2,500 and 5,000 μg/plate), in triplicate, both with and without metabolic activation (S9-mix). The dose range for the main study was determined in a preliminary toxicity test using eight concentrations (3 -5,000 μg/plate) in TA 98 and TA 100 strain. The vehicle 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Toxic effects evident as a reduction in the number of spontaneous revertants, occurred at higher concentrations with and without metabolic activation. The background growth was reduced from 1,000 up to 5,000 μg/plate with and without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Under the conditions of the study, the test substance was considered to be non-mutagenic (Wollny HE, 1997).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/mL respectively) and 30 U/mL heparin.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Dose range finding study:
At 3 h exposure time: 3, 10, 33, 100 and 333 µg/mL culture medium with and without S9-mix.
At 24 and 48 h continuous exposure time: 3, 10, 33, 100, 333 and 1000 µg/mL culture medium without S9-mix


Experiment 1:
Without and with S9-mix: 50, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (3 h exposure time, 24 h fixation time)


Experiment 2:
- Without S9-mix: 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time)
10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix 50, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 10 µg/mL 3 h exposure period ( 24 h fixation time)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.1 µg/mL (48 h exposure), 0.2 (24 h exposure) and 0.5 µg/mL (3 h exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 48 h (Experiment 2 with S9 mix)


SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µ g/mL medium)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED: 1000 cells


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:
A test substance was considered positive (clastogenic) if:
a) A dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations
b) A significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship
A test substance was considered non-clastogenic if:
a) None of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.
Statistics:
One sided, Chi-square test to calculate dose-related statistically significant increase in the number of cells with chromosome aberrations
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: At the 24 and 48 h exposure time, test material was tested beyond the limit of solubility to obtain adequate toxicity data.
- Precipitate of the test material was seen at 333 µg /mL


COMPARISON WITH HISTORICAL CONTROL DATA: Yes, within the laboratory historical control data range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Increased number of polyploid cells in the absence of S9-mix in a dose dependent manner in the first cytogenetic assay indicating potential to inhibit mitotic processes and to induce numerical chromosome aberrations.

The doses selected for scoring of chromosome aberrations:

Without S9-mix: 10, 50 and 100 µg/mL culture medium (24 h exposure time, 24 h fixation time).

50, 100 and 150 µg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix:50, 100 and 300 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Conclusions:
Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro.
Executive summary:

A study was conducted to assess the ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473. In experiment 1, 3 h exposure with a 24 h fixation in the absence and presence of S9-mix (1.8%) were used. In experiment 2, 24 h exposure with a 24 h fixation time and 48 h exposure with a 48 h fixation time in the absence of S9 mix and 3 h exposure with a 48 h fixation in presence of S9 mix were tested. Vehicle control cultures had frequencies of cells with aberrations within the historical control data range. Both of the positive control materials induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence, in either of the two independently repeated experiments. Dose-dependent increase in polyploid cells (absence of S9-mix) in the first cytogenetic assay was noted, indicating the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip CM, 2009).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
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: Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) , 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 h exposure cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium) and for 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction (Rat liver microsomal enzymes prepared from adult male Wistar rats)
Test concentrations with justification for top dose:
First mutagenicity test:
Without S9-mix: 0.1, 0.3, 1, 3, 10, 15, 20, 25, 30, 35, 40 and 45 µg/mL exposure medium
With 8% (v/v) S9-mix: 1, 3, 10, 30, 70, 100, 125, 150, 175, 200, 225 and 250 µg/mL exposure medium

Second mutagenicity test:
Without S9-mix: 0.1, 0.3, 1, 3, 10, 15, 20, 25, 27.5, 30, 32.5 and 35 µg/mL exposure medium
With 12% (v/v) S9-mix: 1, 3, 10, 50, 100, 125, 150, 160, 170, 180, 190 and 200 µg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 15 and 5 µg/mL for a 3 and 24 h treatment period
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3 h (Experiment 2 with S9 mix)
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 d after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CE Day 2) and the mutation frequency (MF).


SELECTION AGENT (mutation assays): Trifluorothymidine


NUMBER OF REPLICATIONS: Two


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more than MF (controls) + 126 in a dose-dependent manner; or
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF (controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 33 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 µg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 333 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

EVALUATION OF THE MUTAGENICITY:
No significant increase in the mutation frequency at the TK locus was observed after treatment with test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material-treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 74% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 77% compared to the total growth of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 95% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 72% compared to the total growth of the solvent controls.

The growth rate over the two-day expression period for cultures treated with DMSO was between 16 and 24 (3 h treatment) and 41 (24 h treatment). Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS in the absence of S9-mix, and by 16 and 9.7-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Conclusions:
Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance in L5178Y mouse lymphoma cells according to OECD Guideline 476. The study was performed in two independent experiments with L5178Y mouse lymphoma cells in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 35 and 175µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. The substance was tested up to cytotoxic levels of 85 and 80 % in the absence and presence of S9-mix, respectively. In the second experiment, the substance was tested up to concentrations of 25 and 200 µg/mL in the absence and presence S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 77% (presence of S9-mix). The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS (absence of S9-mix), and by 16 and 9.7 fold for CP (presence of S9-mix). The test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned appropriately. In both the presence and absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment and this result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip CM, 2009).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the mutagenic potential of the test substance in a reverse mutation (Ames) assay with the tester strains Salmonella typhimurium TA 1535, TA 1537, TA98 and TA100 and Escherichia coli WP2 uvrA according to OECD Guideline 471.

In the dose range finding assay, the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The test substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards in the presence of S9-mix. In strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5000 µg/plate, the number of revertant colonies of this dose level could not be determined. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 µg/plate in the presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains, except in tester strain TA98 in the presence of S9-mix.

In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 µg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 10 to 3330 µg/plate in strain WP2 uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitate on the plates was only observed at the dose level of 3330 μg/plate in the absence of S9-mix. Toxicity was observed in all strains, except in TA1535 and TA1537 in the presence of S9-mix and in WP2 uvrA in the absence and presence of S9-mix. 

Since in the first experiment in strain TA98 and in the second experiment in strains TA1537 and WP2 uvrA no toxicity or precipitate on the plates was observed in the presence of S9-mix, a third mutation experiment was performed with these and strain TA98 in the presence of S9-mix. The substance was tested up to 5000 µg/plate. There was precipitation on the plates at dose levels of 3330 and 5000 μg/plate, so that the bacterial background could not be determined at 3330 and 5000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in strain TA98 in the presence of 5 and 10 %(v/v) S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance is not considered to be mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

An OECD Guideline 471 study was conducted to determine the mutagenic potential of the test substance using a bacterial/microsome test system. Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were exposed to the substance using both the plate incorporation method and pre-incubation method at six concentrations (33, 100, 333, 1,000, 2,500 and 5,000 μg/plate), in triplicate, both with and without metabolic activation (S9-mix). The dose range for the main study was determined in a preliminary toxicity test using eight concentrations (3 -5,000 μg/plate) in TA 98 and TA 100 strain. The vehicle 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Toxic effects evident as a reduction in the number of spontaneous revertants, occurred at higher concentrations with and without metabolic activation. The background growth was reduced from 1,000 up to 5,000 μg/plate with and without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Under the conditions of the study, the test substance was considered to be non-mutagenic (Wollny HE, 1997).

A study was conducted to assess the ability of the test substance to induce chromosome aberrations in cultured peripheral human lymphocytes according to OECD Guideline 473. In experiment 1, 3 h exposure with a 24 h fixation in the absence and presence of S9-mix (1.8%) were used. In experiment 2, 24 h exposure with a 24 h fixation time and 48 h exposure with a 48 h fixation time in the absence of S9 mix and 3 h exposure with a 48 h fixation in presence of S9 mix were tested. Vehicle control cultures had frequencies of cells with aberrations within the historical control data range. Both of the positive control materials induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence, in either of the two independently repeated experiments. Dose-dependent increase in polyploid cells (absence of S9-mix) in the first cytogenetic assay was noted, indicating the potential to inhibit mitotic processes and to induce numerical chromosome aberrations. Under the test conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip CM, 2009).

A study was conducted to determine the mutagenic potential of the test substance in L5178Y mouse lymphoma cells according to OECD Guideline 476. The study was performed in two independent experiments with L5178Y mouse lymphoma cells in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 35 and 175µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. The substance was tested up to cytotoxic levels of 85 and 80 % in the absence and presence of S9-mix, respectively. In the second experiment, the substance was tested up to concentrations of 25 and 200 µg/mL in the absence and presence S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 77% (presence of S9-mix). The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased by 17 and 15-fold for MMS (absence of S9-mix), and by 16 and 9.7 fold for CP (presence of S9-mix). The test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned appropriately. In both the presence and absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment and this result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. Under the test conditions, the substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip CM, 2009).

Justification for classification or non-classification

The test substance was negative in in vitro studies. Therefore, no classification is required for genotoxicity according to CLP (EC 1272/2008) criteria.