Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

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 March 26, 1996 to June 2, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI2004/0994)

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

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)

Constituent 2

Reference substance name:

2,2'-iminodiethanol

EC Number:

203-868-0

EC Name:

2,2'-iminodiethanol

Cas Number:

111-42-2

Molecular formula:

C4H11NO2

IUPAC Name:

2,2'-iminodiethanol

Test material form:

liquid: viscous

Method

Target gene:

No data

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Without metabolic activation: 0, 1.5, 5, 15, 50, 150 and 500 µg/plate
With metabolic activation: 0, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 µg/plate for TA100 and 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine, 5 µg/plate for TA1538

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA1538 and TA98

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 0.5 µg/plate for TA1538 and TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation).

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
The cytotoxicity was rated as follows:
S=sparse background lawn
T=toxic
V=very thin background lawn
C=contaminated
P=precipitate
X=plate unscorable

Evaluation criteria:

The test item was considered positive if there is a dose related increase in the number of revertants or a biologically relevant increase for at least onetest concentration.
The test item was considered negative if the number of induced revertants is less than that of the spontaneous revertants by two fold at each dose level.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Positive controls validity:

not specified

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material exhibited toxicity initially at and above 150 µg/plate to the strain of Salmonella used (TA100).

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic.

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the test substance, C8-18 and C18-unsatd. DEA, according to OECD Guideline 471 and Method B.14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test substance using the Ames plate incorporation method at up to seven dose levels for each bacterial strain, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 1.5 to 500 µg/plate (-S9 mix) and 1.5 to 1500 µg/plate (+S9 mix) in the first experiment. The experiment was repeated on a separate day using a similar dose range to experiment 1, fresh cultures of the bacterial strains and fresh test substance formulations. Extra doses were incorporated into each experiment to allow for test substance toxicity. The vehicle (ethanol) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the S9 mix. The test substance caused a visible reduction in the growth of bacterial lawn to all of the strains of Salmonella tested both with and without metabolic activation. The substance was, therefore, tested up to its toxic limit. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test substance, either with or without metabolic activation. Under the study conditions, the test substance was found to be non-mutagenic (Thompson, 1996).