Poly[oxy(methyl-1,2-ethanediyl)], .alpha.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethyl]-.omega.-[2-[[2,2-dimethyl-3-[(1-oxododecyl)oxy]propylidene]amino]methylethoxy]-

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:

2004-11-30 - 2005-01-14

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:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21st, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 8th, 2000

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

479-940-2

EC Name:

-

Cas Number:

613246-75-6

Molecular formula:

NA: Multi-const. substance

IUPAC Name:

16-(dodecanoyloxy)-2,2,5,8,12,15,15-heptamethyl-7,10-dioxa-4,13-diazahexadeca-3,13-dien-1-yl dodecanoate

Test material form:

liquid

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> 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- -> 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:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (induction with Phenobarbitone and ß-naphthoflavone)

Test concentrations with justification for top dose:

Expermiment I (plate incorporation test) and II (pre-incubation test): 5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate.
Experiment III (confirmatory pre-incubation test): 50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Sika Hardener LJ formed a stable solution or a stable colloid system (an emulsion) in acetone.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 Migrated to IUCLID6: 9AA

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without S9 Migrated to IUCLID6: NaN3

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

TA 98 without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvr A without S9 Migrated to IUCLID6: MMS

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

TA 98, 100, 1535, 1537; E. coli WP2 uvr A with S9

Details on test system and experimental conditions:

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

DURATION
1. plate incorporation
The frozen cultures were thawed at room temperature and a measured amount was used for inoculating the over-night cultures in the assay. 200 µL inoculum was used for each 50 mL of broth. The bacterial strains were grown up in nutrient broth. The cultures were incubated for 10-14 h at 37 °C up to the late exponential or early stationary growth phase (approx. 109 cells/ml) in, a Gyrotory Water Bath Shaker. After the test item was added and mixed and after the solidification, the plates were inverted and incubated at 37°C for at least 48h in the dark. Revertant colonies were counted by hand.

2. preincubation

Histidine- Biotin overlay agar (for Salmonella typhimurium strains) contains per litre:

Agar Bacteriological 3.6 g
NaCl 4.5 g
D-Biotin (F.W. 244.3) 12.4 mg
L-Histidine- HCl H20 (F.W. 209,63) 10.5 mg

The agar solution was sterilised at 121 °C in an autoclave, and the Histidine-Biotin solution (0.5 mM) was sterilised by filtration through a 0.22 µm membrane filter

Tryptophan overlay agar (for Escherichia coli strain) contains per litre:

Agar Bacteriological 3.79 g
NaCl 4.74 g
Nutrient Broth 50.0 ml
L-Tryptophan (F.W. 204.23) 5.0 mg

The agar solution and the nutrient broth was sterilised at 121 °C in an autoclave. The Tryptophan solution (2 mg/mL) was sterilised by filtration through a 0.22 µm membrane filter.

NUMBER OF REPLICATIONS: The concentrations, including the controls, were tested in triplicates.

DETERMINATION OF CYTOTOXICITY

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; 316.205 100.00, 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). The experimental conditions in this pre-experiment were the same as described for the main experiment I (plate incorporation test). Cytotoxic effects of the test item were observed at both examined test strains.

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).

Evalution 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 uvr A the number of reversions is at least three times higher as compared to the spontaneous reversion rate of the solvent control plates.

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In order to investigate the potential of Sika Hardener LJ for its ability to induce gene mutations a plate incorporation test (experiment I) and a pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA.
Inhibitory, toxic effects of the test item were observed in all phases of the study. In experiment I the inhibitory effect of the test item was observed in case of Salmonella typhimurium TA 100, TA 1535 and TA 1537. The revertant colony numbers were reduced compared to the solvent control plates in presence of metabolic activation in the concentration range of 5000.00-158.11 µg/plate in S. typhimurium TA 1537 and in the concentration range of 5000.00-500.00 µg/plate in TA 100 and TA 1535. In absence of metabolic activation the revertant colony numbers were reduced only in case of TA 1537 in the concentration range of 5000.00-500.00 µg/plate. In S. typhimurium TA 100 the appearance of pinpoint colonies (concentration range: 5000.00-1581.14 µg/plate (+S9)) and in TA 1537 reduced background lawn development (5000.00 µg/plate (±S9), 5000.00-1581.14 µg/plate (+S9)) was also observed. 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 in the investigated Salmonella typhimurium strains. The appeared small pinpoint colonies (inall Salmonella typhimurium strains) were also signs of inhibition. The inhibition manifested stronger in the activation part (+S9) of the experiment at higher concentration levels (5000.00-158.11 µg/plate, at TA 1535 5000.00-500.00 µg/plate) with respect to the reduction of revertant colony numbers, but inhibitory effects 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 TA 100, TA 1535 and TA 1537 the toxicity affected even the lowest concentration (5.00 µg/plate). No inhibition was observed in Escherichia coli WP2uvrA.
In the additional confirmatory mutation (experiment III) assay using the preincubation method the results of the experiment II were confirmed. The examined strains Salmonella typhimurium TA 100, TA 1535 and TA 1537 were inhibited without metabolic activation at the concentration range of 50.00-5.00 µg/plate. At the lower concentrations no cytotoxic effects were noted.


TEST-SPECIFIC CONFOUNDING FACTORS: none

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects of the test item were observed at both examined test strains. The revertant colony numbers were slightly reduced compared to the solvent control plates in case of Salmonella typhimurium TA 98 in the concentration range of 5000.00-2500.00 µg/plate, at TA 100 in the concentration range of 5000.00-316.20 µg/plate in presence of metabolic activation system. Beside the reduced revertant colony numbers compared to the solvent control plates reduced background lawn development and appearance of pinpoint colonies was observed as sign of the toxic effect of the test item in Salmonella typhimurium TA 100. The test item did not show mutagenic effect on the examined bacterium strains. The number of the revertant colonies was not significantly enhanced. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon in the concentration range of 5000.00-1000.00 µg/plate.

Applicant's summary and conclusion

Conclusions:

Based on the results of this Ames test, Sika Hardener LJ is considered to be non-mutagenic with or without metabolic activation in bacterial systems.

Executive summary:

Sika Hardener LJ was assessed in an Ames test according to EU method B.13/14 OECD guideline 471. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvr A were testet in a plate incorporation test (experiment I) and a pre-incubation test (experiment II). The test item was tested in two independent experiments (Initial Mutation Assay and Confirmatory Mutation Assay) at seven concentrations each (5000.00; 1581.14; 500.00; 158.11; 50.00; 15.81; 5.00 µg/plate). An additional confirmatory test (experiment III) was carried out, because of the noted toxicity in the experiment II. The examined bacterium strains were; Salmonella typhimurium TA 100, TA 1537 and TA 1535. The experiment was performed in the absence of a post-mitochondrial supernatant (S9). In this confirmatory experiment the preincubation method was also used five different concentrations (50.00; 15.81; 5.00; 1.58; 0.50; 0.16 µg/plate).

No substantial increases in revertant colony numbers of any of the five test strains were observed following treatment with Sika Hardener LJ 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. After 48 hours incubation microdrops (not precipitate) were observed as colloidical chemical phenomenon at the concentrations of 5000.00 and 1581.14 µg/plate. The reported 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 LJ is considered to be non-mutagenic in this bacterial reverse mutation assay.