Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

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:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

EC Number:

285-089-6

EC Name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Cas Number:

85029-63-6

Molecular formula:

not applicable, mixture

IUPAC Name:

Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

Test material form:

liquid

Method

Target gene:

The histidine dependent strains are derived from S. typhimurium strain LT2 throught a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
Strain WP2 and its derivatives all carry the same defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent (Trp+) mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen which substitute one base for another. Additionally, the uvrA derivative is deficient in the DNA repair process (excision repair damage). Such a repair-deficient strain may be more readily mutated by agents.
When summarised the mutations on the TA strains and the E. coli strain, used in this study can be described as follows:

SALMONELLA TYPHIMURIUM:
-Strain TA 1537; Genotype: his C 3076; rfa-; uvrB-: Type of mutations indicated: frame shift mutations
-Strain TA 98; Genotype: his D 3052; rfa-; uvrB-; R-factor Type of mutations indicated: frame shift mutations
-Strain TA 1535; Genotype: his G 46; rfa-; uvrB-: Type of mutations indicated: base-pair substitutions
-Strain TA 100; Genotype: his G 46; rfa-; uvrB-; R-factor Type of mutations indicated: base-pair substitutions

ESCHERICHIA COLI
-Strain WP2 uvrA; Genotype: trp-; uvrA-: Escherichia coli: base-pair substitutions and others

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

Test concentrations with justification for top dose:

In the pre-experiment the concentration rnge of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
The test item precipitated in the overlay agar in experiment I with and without metabolic activation at 5000, except in strain TA 1537 (without metabolic activation) and WP2 uvrA with metabolic activation precipitation was observed at 2500 - 5000 µg/plate.
The undissolved particles had no influence on the data recording.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Negative Controls: Concurrent untreated and solvent controls were performed.

S9 (Preparation by RCC-CCR): Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i. p. (Desitin; D-22335 Hamburg) and beta-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution followed by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week.
The protein concentration in the S9 preparation was 34.5 mg/mL (lot no. R 300404).

S9 Mix:
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Rationale for test conditions:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.

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:

no 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

Remarks:

at 2500 - 5000 µg/plate with and without S9 mix in Experiment I and at 33 - 1000 µg/plate without S9 mix and 2500 µg/plate with S 9 mix in Experiment II

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

Remarks:

at 5000 µg/plate with S9 mix in Experiment I and II

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

Remarks:

at 1000 - 5000 µg/plate without S9 mix and at 2500 - 5000 µg/plate with S9 mix in Experiment I, at 333 - 5000 µg/plate without S9 mix and 2500 - 5000 µg/plate with S9 mix in Experiment II

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:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix in Experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The test item was assessed for its poetntail to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	/	/
TA 1537	2500 - 5000	/	1000	2500 - 5000
TA 98	5000	2500 - 5000	5000	2500 - 5000
TA 100	333 - 5000	1000 - 5000	1000 - 5000	/
WP2 uvrA	5000	1000 - 5000	/	/

/ = no reduced background growth

Toxic effects, evident as a reduction in the number of revertants, were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	/
TA 1537	2500 - 5000	2500 - 5000	33 - 1000	2500
TA 98	/	5000	/	5000
TA 100	1000 - 5000	2500 - 5000	333 - 5000	2500 - 5000
WP2 uvrA	5000	5000	/	/

/ = no toxic effects evident as a reduction in the number of revertants

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In strain WP2 uvrA of the second experiment without metabolic activation the historical range of positive controls was just not reached. This minor effect was judged to represent fluctuations. The threshold of two times the corresponding solvent control was exceeded by far (factor of 6.5), so the test was considered valid.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore the substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in three independent experiments two with and without liver microsomal activation. A third experiment had to be performed, since in experiment I with metabolic activation in strain TA 1537 no bacteria were applicated onto the plates (reported as part of experiment I). Each concetration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	5000	5000	/	/
TA 1537	2500 - 5000	/	1000	2500 - 5000
TA 98	5000	2500 - 5000	5000	2500 - 5000
TA 100	333 - 5000	1000 - 5000	1000 - 5000	/
WP2 uvrA	5000	1000 - 5000	/	/

/ = no reduced background level

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations in strains TA 1537, TA 98, 100 and WP2 uvrA.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, meither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.