Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the conditions of the study the test material was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2018 to 25 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Preparation of test material
No correction was made for the purity/composition of the test material.
A solubility test was performed based on visual assessment. The test material formed a light brown homogenous suspension in ethanol at 50 mg/mL. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
Test material concentrations were used within 2.5 hours after preparation.
Any residual volumes were discarded.
Target gene:
Salmonella typhimurium
TA1537: his C 3076; (frame shift mutations)
TA98: his D 3052; R-factor (frame shift mutations)
TA1535: his G 46; (base-pair mutations)
TA100: his G 46; R-factor (base-pair mutations)

Escherichia coli
WP2 uvrA: trp-; (base-pair substitution)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- The Salmonella typhimurium strains are checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- Stock cultures of the five strains were stored in liquid nitrogen (-196 °C).
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment. The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
- Stock cultures of the five strains were stored in liquid nitrogen (-196 °C).
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
- Dose range-finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA100 and WP2uvrA; with and without metabolic activation)

- First mutation experiment: 5.4, 17, 52, 164, 512 and 1600 μg/plate (TA1535, TA1537 and TA98; with and without metabolic activation - 5% v/v S9-mix)
- Second mutation experiment: 48, 86, 154, 275, 492, 878 µg/plate (TA1535, TA1537, TA98, TA100 and WP2uvrA; with and without metabolic activation - 10% v/v S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY 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 vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at The Test Facility.
b) 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 extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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 follow up experiment.
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive 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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE-FINDING TEST/ FIRST MUTATION EXPERIMENT
The test material was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 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 first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate.

- Precipitate
Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 164 µg/plate and upwards and at 512 µg/plate and above at the end of the incubation period.

- Toxicity
To determine the toxicity of, 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 the first mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants up to the dose level of 1600 μg/plate. Since the test material precipitated heavily on the plates at 5000 µg/plate, the number of revertants and the bacterial background lawn at this dose level could not be determined.

- Mutagenicity
No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

SECOND MUTATION EXPERIMENT
To obtain more information about the possible mutagenicity of the test material, a second mutation experiment was performed in the absence and presence of 10 % (v/v) S9-mix. Based on the results of the first mutation assay, the test material was tested up to the dose level of 878 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

- Precipitate
Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 86 µg/plate and above and at 275 µg/plate and above at the end of the incubation period.

- Toxicity
In the second mutation assay, 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 all tester strains in the absence and presence of S9-mix.
In tester strain TA100, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at 275 µg/plate in the presence of S9-mix. However, since no dose-relationship was observed and the reduction was less than 20 % compared to the concurrent vehicle control, this reduction is not considered to be caused by toxicity of the test material. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

- Mutagenicity
In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

Table 1: Experiment 1 - mutagenic response of the test material

Dose (µg/ plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium

TA 1535

TA1537

TA98

Without S9-mix

Positive control

1129

1228

860

Solvent control

6

5

15

5.4

8

4

17

17

9

4

15

52

10

5

21

164

4

NP

3

NP

15

NP

512

10

SP

6

SP

17

SP

1600

7

n MP

5

n MP

13

n MP

With S9-mix*

Positive control

262

217

1120

Solvent control

12

6

27

5.4

8

6

17

17

6

4

22

52

9

6

19

164

9

NP

5

NP

25

NP

512

10

SP

8

SP

25

SP

1600

9

n MP

5

n MP

20

n MP

 * plate incorporation assay (5 % S9)

MP: moderate precipitate

NP: No precipitate

SP: Slight precipitate

n: normal bacterial background lawn

Table 2: Experiment 2 - mutagenic response of the test material

Dose (µg/ plate)

Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and one Escherichia coli strain

TA 1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

1130

752

958

970

1608

Solvent control

7

3

19

93

18

48

13

5

23

106

30

86

6

7

15

109

21

154

8

NP

3

NP

20

NP

92

NP

21

NP

275

10

SP

5

SP

15

SP

107

SP

17

SP

492

8

SP

3

SP

20

SP

110

SP

23

SP

878

6

n MP

3

n MP

13

n MP

110

n MP

20

n MP

With S9-mix*

Positive control

262

100

562

473

293

Solvent control

13

6

18

61

24

48

7

5

21

60

28

86

13

7

23

70

19

154

10

NP

4

NP

23

NP

61

NP

21

NP

275

11

SP

6

SP

19

SP

58

SP

21

SP

492

10

SP

4

SP

20

SP

61

SP

22

SP

878

13

n SP

4

n SP

24

n SP

76

n SP

31

n SP

* plate incorporation assay (10 % S9)

MP: moderate precipitate

NP: No precipitate

SP: Slight precipitate

n: normal bacterial background lawn

Conclusions:
All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two experiments.
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.
In conclusion, based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions.

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 512 μg/plate and upwards. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants up to the dose level of 1600 μg/plate. Since the test material precipitated heavily on the plates at 5000 µg/plate, the number of revertants and the bacterial background lawn at this dose level could not be determined. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 µg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at dose levels of 512 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 48 to 878 µg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 275 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.  

The test material 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up 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.

In conclusion, based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.   

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

Additional information

The mutagenicity of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 512 μg/plate and upwards. There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants up to the dose level of 1600 μg/plate. Since the test material precipitated heavily on the plates at 5000 µg/plate, the number of revertants and the bacterial background lawn at this dose level could not be determined. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 5.4 to 1600 µg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at dose levels of 512 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 48 to 878 µg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 275 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.  

The test material 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up 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.

In conclusion, based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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.