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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 6, 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: viscous liquid
Details on test material:
- Name of test material (as cited in study report):Amides, C8-18 and C18 unsatd., N,N bis(hydroxyethyl)
- Physical state: Clear yellow viscous liquid
- Analytical purity: 100%
- Lot/batch No.: 7568115TBT6
- Storage condition of test material: At room temperature in the dark
- Other: If the test substance is solidified, gentle warming up to approximately 35˚C for as long as it takes to re-liquefy

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/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 with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate.The results are shown in Table 3 and Table 4 of the study report.  

Precipitate

Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

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. The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in Table1.

Table 1: Toxicity of the test substance in the dose range finding test/first mutation 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          extreme          microcolonies

1000          extreme         microcolonies

 1000        extreme         microcolonies

TA1537

 100          normal               moderate

 333          extreme         microcolonies

1000          extreme        microcolonies

1000          extreme         microcolonies

TA98

 333          moderate          extreme

1000          extreme        microcolonies

1000          extreme         microcolonies

TA100

100-333     moderate          extreme

1000-5000  extreme         microcolonies

 333          moderate          extreme

1000-5000  extreme         microcolonies

WP2 uvrA

5000          normal               -1

5000          normal             slight     

-1  No reduction in the number of revertant colonies

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 and with S9-mix: 3, 10, 33, 100, 333 and 1000 μg/plate

WP2 uvrA: Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

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

Precipitate

Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period.

Toxicity

The reduction of the bacterial background lawn and the reduction in the number of revertants is presented inTable 2.

Table 2: Toxicity of the test substance in the second mutation 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      extreme           microcolonies

1000     extreme           microcolonies

1000          extreme              microcolonies

TA1537

 333      absent             complete

1000     absent             complete

 333          slight                 -1

1000          extreme              microcolonies

TA98

 333      slight                 extreme

1000     extreme           microcolonies

1000          moderate            extreme

TA100

 100      normal             moderate

 333      extreme           microcolonies

1000     extreme           microcolonies

1000          extreme              microcolonies

WP2 uvrA

5000     normal                     -2

5000           normal                  -2

-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.

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 atructurally related read-across substance amides, C8-18(even numbered) and C18 unsat., N,N bis(hydroxyethyl) 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 with Salmomenlla typhimurium strain TA100 and Escherichia coli strain WP2 uvrA, the substance was tested up to concentrations of 5,000 µg/plate in triplicate in both the presence and absence of 5% (v/v) S9-mix. No precipitation was observed on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100, 333, 1,000, 3,330 and 5,000 µg/plate in the absence of 5% (v/v) S9-mix and at 333, 1,000, 3,330 and 5,000 µg/plate in the presence of 5% (v/v) S9-mix. InEscherichia colistrain WP2 uvrA, toxicity was observed at the dose level of 5,000 µg/plate in the presence of 5% (v/v) S9-mix. Based on the preliminary test, the substance was evaluated at concentrations of 3, 10, 33, 100, 333 and 1,000 µg/plate in the presence and absence of 5% (v/v) S9-mix in strains TA1535, TA1537 and TA98 in the first mutation assay. Toxicity was observed in all strains. In the second mutation assay, the substance was tested up to concentrations of 1,000 µg/plate in the presence and absence of 5% (v/v) S9-mix in strains TA 100, TA1535, TA1537 and TA98 and up to concentrations of 5,000 µg/plate in strain WP2 uvrA. Toxicity was observed in all strains except WP2 uvrA. 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 strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independent repeat. In this study, 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 was concluded that the test substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.