N,N-bis(2-hydroxyethyl)undec-10-enamide

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:

From February 28, 2017 to March 23, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

N,N-bis(2-hydroxyethyl)undec-10-enamide

EC Number:

262-114-9

EC Name:

N,N-bis(2-hydroxyethyl)undec-10-enamide

Cas Number:

60239-68-1

Molecular formula:

C15H29NO3

IUPAC Name:

N,N-bis(2-hydroxyethyl)undec-10-enamide

Test material form:

liquid

Details on test material:

The test substance is the reaction product of 10-undecenoicacid methyl ester with diethanolamine (DEA). The methanol formed in the process is removed by distillation. The final product is a clear liquid at 20 °C and 101.3 kPa.

Specific details on test material used for the study:

Batch no.: S016419544
Purity: 90.60%
Apearance: yellow, clear liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

microsomal preparation derived from Aroclor 1254-induced rat liver (S9-mix)

Test concentrations with justification for top dose:

Six concentrations ranging from 3.16 to 1000 μg/plate (3.16, 10.0, 31.6, 100, 316 and 1000 μg per plate) based on a preliminary test where cytotoxicity was observed from 1000 µg/plate and onwards.

Vehicle / solvent:

DMSO (as well as ethanol and water)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

- The potential of the test substance to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
(Plates: 3 per concentration and experiment; the final cell density was approximately 10E08 - 10E09 cells/mL.)

- The test substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at a concentration of 1000 μg/plate in both experiments. Hence, 1000 μg per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

- Main study
Six concentrations ranging from 3.16 to 1000 μg test substance/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Rationale for test conditions:

Preliminary test

Evaluation criteria:

Bacteria colonies were counted employing the Biosys Biocount 5000 system. Occurrence of test substance precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.

Statistics:

p ≤ 0.05, U-test according to MANN and WHITNEY compared to the solvent control
(concentration-related increase over the range tested in the number of the revertants per plate, reproducibility)

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella t. TA98, TA00, TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest dose of 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Cytotoxicity
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 μg/plate in all test strains.
- Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to the cytotoxic concentration of 1000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
- The positive control substances showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce any increase in the number of revertants and was therefore considered not mutagenic in Salmonella typhimurium.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the tes substance according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. The potential of the test substance to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test substance was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg/plate were tested. Pronounced cytotoxicity was noted starting at a concentration of 1000 μg/plate in both experiments. Hence, 1000 μg per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. In the main study, six concentrations ranging from 3.16 to 1000 μg test substance/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 μg/plate in all test strains. No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to the cytotoxic concentration of 1000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test). The positive control substances showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data. Under the study conditions, the test substance did not induce any increase in the number of revertants and was therefore considered not mutagenic in Salmonella typhimurium (Spruth, 2017).