Amides, coco, N,N-bis(hydroxyethyl); C10-12 and C18-unsatd. DEA

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 6, 2014 to March 7, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

EC Number:

931-329-6

Cas Number:

68155-07-7

Molecular formula:

The alkyl chain length of the amide ranges between 8 and 18 carbon atoms

IUPAC Name:

Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Saline

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

TA1535; concentration/plate 5µg

Controls 3

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

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98; concentration/plate 10µg

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

TA100; concentration/plate 650µg

Controls 6

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

Controls 7

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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Remarks on result:

other: no mutagenic potential

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:

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

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance, C8-18 and C18-unsatd. DEA, 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 according to OECD Guideline 471, in compliance with GLP. In the dose range finding study, 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 did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and upwards in the absence of metabolic activation (S9-mix) and at dose levels of 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, toxicity was only observed at the dose level of 5000 μg/plate in the presence of S9-mix. Based on the results of the dose range finding study, the substance was tested in the first mutation assay at a concentration range of 3 to 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In the second mutation assay, the substance was tested up to concentrations of 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA100, TA1535, TA1537 and TA98 and up to concentrations of 5000 µg/plate in tester strain WP2 uvrA. Toxicity was observed in all tester strains, except in tester strain 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 tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. 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. Under the conditions of the study, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2014).