Dodecanoic acid, 1,1'-[1,6-hexanediylbis[nitrilo(2,2-dimethyl-1-propanyl-3-ylidene)]] ester

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:

2004-11-26 to 2005-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1996

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:

-

EC Number:

479-930-8

EC Name:

-

Cas Number:

613222-52-9

Molecular formula:

C40H76N2O4

IUPAC Name:

(3E)-3-({6-[(E)-[3-(dodecanoyloxy)-2,2-dimethylpropylidene]amino]hexyl}imino)-2,2-dimethylpropyl dodecanoate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- to his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– to trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S9-mix from rats induced with Phenobarbitone (PB) and ß-naphthoflavone (BNF)

Test concentrations with justification for top dose:

5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate.
In case of the tester strains Salmonella typhimurium TA100, TA 1535 and TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) in experiment II.
Therefore, an additional (third) confirmatory test was carried out with Salmonella typhimurium TA 100, TA 1535 and TA 1537 and the following concentrations were tested: 50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate.

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate; MMS

Remarks:

E. coli WP2 uvrA without S-9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

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

Remarks:

S. typhimurium: TA 98 without S-9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium: TA 100; TA 1535 without S-9 Migrated to IUCLID6: NaN3

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

S. typhimurium TA 1537 without S-9 Migrated to IUCLID6: 9-AA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; 2-AA

Remarks:

S. typhimurium: TA 100; TA 98; TA 1535; TA 1537 and E. coli WP2 uvrA with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number

Evaluation criteria:

Evaluation of experimental data
The colony numbers on the control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values were used for this calculation).

Evaluation of Results
The test is considered acceptable if for each strain:
– the bacteria demonstrate their typical responses to crystal violet and ampicillin
– the control plates without S9 mix are within the historical control data range
– corresponding background growth on both negative control and test plates occurs
–the positive controls show a distinct enhancement over the control plate

A test item is considered mutagenic if:
– a dose–related increase in the number of revertants occur and/or
– a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

A biologically relevant increase is described as follows:
– if in strain TA 100 the number of reversions is at least twice as high when compared to the spontaneous reversion rate of the solvent control plates,
– if in strains TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results; a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

The colony numbers on the control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The Mutation Factor was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values were used for this calculation).

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate.

RANGE-FINDING/SCREENING STUDIES: Inhibitory, toxic effects of the test item were observed in all phases of the study. In experiment I the inhibitory effect was observed in all Salmonella typhimurium strains. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in S. typhimurium TA 98 and TA 1535 at the highest concentration level of 5000.00 g/plate, in S. typhimurium TA 100 in the concentration range of 5000.00-500.00 µg/plate, and in S. typhimurium TA 1537 in the concentration range of 5000.00-1581.14 µg/plate. The revertant colony numbers were also reduced without metabolic activation: In Salmonella typhimurium TA 100 in the concentrations of 5000.00 and 1581.14 µg/plate and in TA 1537 in the concentration range of 5000.00-158.11 µg/plate. Background lawn reduction was detected in TA 100 (+S9; at 5000.00 and 1581.14 µg/plate), in TA 1535 (+S9; at 5000.00 µg/plate) and in TA 1537 (+/-S9; at 5000.00, 1581.14 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA. In the experiment II using the pre-incubation method, the inhibitory effect manifested much stronger. The pre-incubation method is more sensitive than the plate incorporation assay. The revertant colony numbers compared to the solvent control plates as well as the background lawn development were reduced and small pinpoint colonies appeared in the investigated Salmonella typhimurium strains.

The inhibition manifested stronger in the activation part (+S9) of the experiment at higher concentration levels (5000.00-158.11 µg/plate) with respect to the reduction of revertant colony numbers, but the inhibitory effect could be observed down to lower concentration levels in the non-activation part of the experiment in all cases. In the test strains Salmonella typhimurium TA100, TA 1535 and TA1537 the toxicity affected even the lowest concentration (5.00 µg/plate) while in case of TA 98 it was observable down to the concentration preceding the lowest concentration (15.81 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA.

In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. The examined strains were inhibited without metabolic activation at the concentration range of 50.00-5.00 µg/plate. The inhibition manifested in reduction of revertant colony numbers, in reduction of background lawn development and in appearance of pinpoint colonies. At the lower concentrations no cytotoxic effects were noted.

HISTORICAL CONTROL DATA:
- Historical control values for spontaneous revertants per plate are as follows: (-S9) Salmonella typhimurium TA 98: 15-60, TA 100: 75-200, TA 1537: 3-30, TA 1535: 3-28 and Escherichia coli WP2 uvrA: 8-50.

Applicant's summary and conclusion

Conclusions:

Sika Hardener LH is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

SIKA Hardener LH was assessed in an Ames test according to EU method B.13/14, OECD guideline 471 and EPA OPPTS 870.5100. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were tested in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). Two independent experiments were run (Initial Mutation Assay and Confirmatory Mutation Assay) up to limit or cytotoxic concentrations, with and without metabolic activation (S9 mix). All assays, including controls, were tested in triplicate.

Inhibitory effects of treatment were observed in all phases of the study. In the experiment I the inhibitory effect appeared in reduction of spontaneous rate compared to the solvent control values (Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537), and in background lawn reduction (Salmonella typhimurium TA 100, TA 1535 and TA 1537). In the experiment II revertant colony numbers compared to the solvent control plates as well as the background lawn development were reduced and small pinpoint colonies appeared in the investigated Salmonella typhimurium strains. Reduction of revertant colony numbers was pronounced at high concentration levels (5000.00-158.11 µg/plate) with metabolic activation, but observed down to lower concentrations in non-activated treatments. In test strains Salmonella typhimurium TA100, TA 1535 and TA1537 cytotoxic effects were observed even at lowest concentrations (5.00 µg/plate) and for TA 98 observed down to 15.81 µg/plate. No cytotoxic effects were observed in Escherichia coli WP2uvrA. In the additional confirmatory mutation (experiment III) assay using the pre-incubation method the results of the experiment II were confirmed. Cytotoxic effects were observed at concentrations of 50.00-5.00 µg/plate without metabolic activation. At the lower concentrations no cytotoxic effects were noted.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with SIKA Hardener LH at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the solvent control values were observed in all experimental phases of the study. However, there was also no tendency of higher mutation rates with increasing concentrations in the range beyond the generally acknowledged border of biological relevance in the performed experiment. The revertant colony numbers of solvent control plates without S9 mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

The data of this mutagenicity assay show that 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, SIKA Hardener LH was considered non-mutagenic in this bacterial reverse mutation assay.