Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-hydro-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-, ether with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (3:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-11-26 to 2005-01-16

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

cited as : 2000/32/EG, dated 2000-06-08

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Different oligomers of 1,1,1-tris-(omega-(2,2-dimethyl-3-lauroyloxypropylidene-amino)-poly(oxy(methyl-1,2-ethanediyl))-methyl)-propane

Molecular formula:

C57H107N3O6 × [C3H6O](x+y+z); (x+y+z) = ca. 5.3 (average)

IUPAC Name:

Different oligomers of 1,1,1-tris-(omega-(2,2-dimethyl-3-lauroyloxypropylidene-amino)-poly(oxy(methyl-1,2-ethanediyl))-methyl)-propane

Constituent 2

Reference substance name:

Different oligomers of 1,1-Bis(omega-(2,2-dimethyl-3-lauroyloxy-propylidene-amino))-poly(oxy(methyl-1,2-ethanediyl))methyl)-1-(omega-hydroxy-poly(oxy(methyl-1,2-ethandiyl))-methyl)propane

Molecular formula:

C40H76N2O5 × [C3H6O](x+y+z); (x+y+z) = ca. 5.3 (average)

IUPAC Name:

Different oligomers of 1,1-Bis(omega-(2,2-dimethyl-3-lauroyloxy-propylidene-amino))-poly(oxy(methyl-1,2-ethanediyl))methyl)-1-(omega-hydroxy-poly(oxy(methyl-1,2-ethandiyl))-methyl)propane

Constituent 3

Reference substance name:

Different oligomers of 1-(omega-(2,2-dimethyl-3-lauroyloxy-propylidene-amino)-poly(oxy(methyl-1,2-ethanediyl))-methyl)-1-bis-(omega-hydroxy-poly(oxy(methyl-1,2-ethanediyl))-methyl)-propane

Molecular formula:

C23H45N1O4 × [C3H6O](x+y+z); (x+y+z) = ca. 5.3 (average)

IUPAC Name:

Different oligomers of 1-(omega-(2,2-dimethyl-3-lauroyloxy-propylidene-amino)-poly(oxy(methyl-1,2-ethanediyl))-methyl)-1-bis-(omega-hydroxy-poly(oxy(methyl-1,2-ethanediyl))-methyl)-propane

Constituent 4

Reference substance name:

"unknown" (oligomers)

Molecular formula:

no data

IUPAC Name:

"unknown" (oligomers)

Constituent 5

Reference substance name:

-

EC Number:

468-880-2

EC Name:

-

Cas Number:

102985-93-3

Molecular formula:

C17H32O3

IUPAC Name:

2,2-dimethyl-3-oxopropyl 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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 5000; 1581.14; 500; 158.11; 50; 15.81; 5.00 µg/plate.
Concentration range in the main test (without metabolic activation): 5000; 1581.14; 500; 158.11; 50; 15.81; 5.00 µg/plate

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

S. typhimurium: TA 1537

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

Remarks:

S. typhimurium: TA 100; TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

S. typhimurium: TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

S. typhimurium: TA 100; TA 98; TA 1535; TA 1537 an d E. coli WP2 uvrA

Details on test system and experimental conditions:

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

The bacterial strains were cultured overnight in Nutrient Broth No. 2. The next day molten top agar was prepared and kept at 45 °C until further use. The test item and other components were prepared freshly and added to the overlay (45 °C). Bacteria were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.

For activation part of this study instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of a non-activation and an activation test conditions each of them with negative and positive controls. For the pre-incubation method before the overlaying the test item, the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes, provided the direct contact between bacteria and the test item. These tubes were gently mixed and incubated for 20 min at 37 °C by using a shaker. After the incubation the content of the tubes were added to the molten top agar prior to pouring onto the surface of minimal agar plates. For activation part of this study instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of a non-activation and an activation test conditions each of them with negative and positive controls. After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

- NUMBER OF REPLICATIONS: 3 replicates per control or concentration levels.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Inhibitory, toxic effect of the test item was observed. The inhibitory effect of the test item manifested in reduction of revertant colony numbers compared to the spontaneous revertant colony numbers of the solvent control plates, in reduced background lawn development and in appearance of small pinpoint colonies.

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with Sika Hardener LTJ at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
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.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains TA 98 and TA 100 in a pre-experiment. 8 concentrations (5000.00; 2500.00; 1000.00; 316.20, 100.00, 31.62, 10.00 and 3.162 µg/plate) were tested for toxicity and mutation induction with each 3 plates in the presence and absence of metabolic activation system (S9). Cytotoxic effects of the test item were observed at both examined test strains. The test item did not show mutagenic effect on the examined bacterium strains. The number of the revertant colonies was not enhanced.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that,the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Sika Hardener LTJ is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A study was conducted according to OECD TG 417 and Directive 2000/32/EG. The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at several concentrations. Each assay was conducted with and without metabolic activation (S9 mix). The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in experiment I based on the results obtained in the pre-experiment on toxicity:

5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate Inhibitory, toxic effect of the test item was observed in both experiments (I and II). The inhibitory effect of the test item manifested in reduction of revertant colony numbers compared to the spontaneous revertant colony numbers of the solvent control plates, in reduced background lawn development and in appearance of small pinpoint colonies. No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with Sika Hardener LTJ (VP) at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.

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. Therefore, Sika Hardener LTJ (VP) is considered to be non-mutagenic in this bacterial reverse mutation assay.