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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
February 17, 2014 to March 7, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 471 (Bacterial Reverse Mutation Assay)/EU Method B14 and is GLP compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: pale cream off white flakes
Details on test material:
- Name of test material (as cited in study report):Amides, C8-18(even numbered) and C18 unsatd., N-(hydroxyethyl)
- Physical state: Pale cream off white flakes
- Analytical purity: 100%
- Lot/batch No.: 7582928
- Storage condition of test material: At room temperature in the dark

Method

Species / strainopen allclose all
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
Controlsopen allclose all
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.


Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Dose range finding test/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 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 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: Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 666 and 1000 µg/plate and no precipitate was observed at the end of the incubation period. Except in tester strain TA98 where no precipitate 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 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 revertants of this dose level could not be determined. The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains are presented in Table 1.

Table1: Toxicity of Amides, C8-18(even numbered) and C18 unsatd., N-(hydroxyethyl) 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

TA1535

333          moderate                  -2

666          extreme          microcolonies

 333          slight                         -1

 666          slight                         -2

1000        moderate                   -2

TA1537

333          moderate                  -1

666          extreme          microcolonies

 333          slight                         -1

 666          slight                         -2

1000        moderate             extreme

TA98

666           normal               extreme

1000         normal                       -2

TA100

 333         slight                 moderate

1000         extreme          microcolonies

3330         absent              complete

5000         absent                      -3

1000         moderate          extreme

3330         absent             complete

5000         absent                      -3

-1  No reduction in the number of revertant colonies

-2  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

-3  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, TA98, TA100: Without S9-mix: 3, 10, 33, 100, 333 and 666 μg/plate and with S9-mix: 10, 33, 100, 333, and 1000 μg/plate WP2 uvrA: Without and with S9-mix: 10, 33, 100, 333, 1000 and 3300 µg/plate The results are shown in Table 5 of the study report. Precipitate Precipitation of the test substance on the plates was only observed in tester strain WP2 uvrA at the start of the incubation period 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. At the end of the incubation period, precipitation on the plates was only observed at 3330 µg/plate in the absence 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. Table2: Toxicity of the test substance in second 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

TA1535

333          moderate                  -2

666          extreme             absent

1000        moderate                   -1

TA1537

333          slight                  moderate

666          extreme           microcolonies

1000        normal                       -2

TA98

666           moderate          extreme

1000         normal             moderate

TA100

 100          slight                 moderate

 333         moderate           extreme

1000         extreme          microcolonies

 333         normal             moderate

 666         moderate         extreme

1000         moderate         extreme

-1  No reduction in the number of revertant colonies

-2  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 and in the second experiment in the tester strains TA1537 and WP2 uvrA no toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore a third mutation experiment was performed with these strains and tester strain TA98 in the presence of S9-mix at a concentration range of 333 to 5000 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 concentrations of 3330 and 5000 µg/plate. Toxicity Due to precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 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 tester strain TA98 in the presence of 5 and 10 %(v/v) S9-mix. Mutagenicity In the third mutation assay, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Applicant's summary and conclusion

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

The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the E.coli reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance in the Salmonella typhimurium reverse mutation assay (strains TA 1535, TA 1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay strain WP2 uvrA. The test was conducted in the presence and absence of metabolic activation (S9 -mix). The study was conducted according to the most recent OECD and EC guidelines.

In the dose range finding test, the test 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 tester 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 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 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 test 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 tester 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 tester strains, except in tester strain 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 tester strain TA98 and in the second experiment in tester 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 strains and tester strain TA98 in the presence of S9-mix. The test substance was tested up to 5000 µg/plate. The test substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Due to the precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 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 tester 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.

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. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.