Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

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:

1st December 2003 to 20th January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted to GLP. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: CPMP/ICH/141/95

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: CPMP/ICH/174/95

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: USA EPA 712-C-98-247

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

EC Number:

269-358-5

EC Name:

Lactic acid, compound with 3-[2-(dimethylamino)ethyl] 1-(2-ethylhexyl) toluene-2,4-dicarbamate (1:1)

Cas Number:

68227-46-3

Molecular formula:

C21H35N3O4.C3H6O3

IUPAC Name:

2-hydroxypropanoic acid 2-(dimethylamino)ethyl N-[5-({[(2-ethylhexyl)oxy]carbonyl}amino)-2-methylphenyl]carbamate

Details on test material:

- Name of test material (as cited in study report): WRS-2390TX
- Physical state: Vicous liquid
- Analytical purity: 71.2% in water
- Expiration date of the lot/batch: 15th November 2004
- Storage condition of test material: The test item was stored at ambient laboratory temperature in the dark for the duration of the study.

Method

Target gene:

nda

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The dose levels for the first mutation assay, based on the results of the toxicity test were:
1, 3.3, 10, 33.3, 100 and 333 μg per plate.

The dose levels for the second mutation assay in the presence of S9 mix were:
1, 3.3, 10, 33.3, 100 and 333 μg per plate.

The dose levels for the second mutation assay in the absence of S9 mix were:
0.5, 1.7, 5, 17, 50 and 167 μg per plate.

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for the assay in the presence of S9 mix, sodium azide (TA1535 and TA-100), 9-aminoacridine (TA-1537), 2-nitrofluorene (TA-98)and N-ethyl-N-nitro-N-nitrosoguanidine (E.coli) for the assay in the absence of S9.

Details on test system and experimental conditions:

Aroclor-1254 induced S9 enzymes (the supernatant of the post-mitochondrial 9000 g fraction) were prepared from the livers of adult, male Fischer rats.
S9 was stored in sterile plastic tubes immersed in liquid nitrogen (ca -196°C).

The enzymic activity of each batch of S9 was characterised by testing selected pre-mutagens in an Ames test with S. typhimurium TA 1538. S9 batches used must also have shown, within each test, a satisfactory mutagenic response in bacteria treated with 2-aminoanthracene.

Ice-cold 0.05 M phosphate buffer, pH 7.4, was added to the following pre-weighed reagents to give final concentrations in S9 mix of:
NADP di-Na salt 4 mM (= 3.150 mg.mL-1)
Glucose-6-phosphate di-Na salt 25 mM (= 7.603 mg.mL-1)
MgCl2.6H2O 8 mM (= 1.626 mg.mL-1)
KCl 33 mM (= 2.460 mg.mL-1)
This solution was immediately filter-sterilised by passage through a 0.45 μm disposable filter assembly and mixed 9:1 (v/v) with the S9.

Preparation of Bacteria:
Samples of each strain were grown by culturing for 16 h at ca 37°C in nutrient broth (25 g Oxoid Nutrient Broth No. 2 per litre). These cultures were kept for up to 4 days at ca 4°C to allow relevant checks to be performed but fresh cultures were used for the experiments.

Preparation of the Assay Plates:
Diluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was sterilised by autoclaving. L-histidine and biotin solutions, and L-tryptophan solutions were sterilised by filtration.
For use with the S. typhimurium strains, sterile 1.0 mM L-histidine.HCl/1.0 mM biotin solution was added, 5 mL per 100 mL of soft agar. For E. coli WP2uvrA, 1.0 mL of 1.35 mM L-tryptophan was added per 100 mL agar. The agars (with additions) were thoroughly mixed prior to use and maintained in a water bath at ca 45°C.
In the course of testing, 2 methods of treatment were performed to extend the range of conditions within the assay. The tests performed were the Direct Plate method and the Pre-incubation method.


Two independent mutation tests were conducted each using 5 bacterial strains. Triplicate plates were prepared for each bacterial strain and dose levels in boththe presence and absence of metabolic activation (S9).
The Direct Plate Method:
Volumes of soft agar (2 mL) were dispensed into small sterile tubes. To this, 0.5 mL of S9 mix or 0.05 M phosphate buffer, pH 7.4 were added, followed by 0.1 mL of bacteria. The solvent or test solution (0.1 mL) was added last.
The tube contents, which were continually cooling, were mixed and poured onto minimal medium plates, prepared in-house. These plates contained 20 mL of 1.5% purified agar, in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and incubated at ca 37°C for 2 or 3 days.

The Pre-incubation Method:
Volumes of S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) were dispensed into small sterile tubes. This was followed by 0.1 mL of bacteria per tube and, finally, the solvent or test solution (0.1 mL per tube). The tube tops were then screwed on tightly and the tubes placed in a shaking incubator at ca 37°C for 20 min. After this, the tube tops were removed and 2 mL of soft agar added to each tube. The tube contents, which were continually cooling, were mixed and then poured onto agar plates, as above. When the soft agar had set, the plates were inverted and incubated at ca 37°C for 2 or 3 days.
After incubation, the colonies were counted using a Cardinal Colony Counter (Perceptive Instruments, UK) set at maximum sensitivity, i.e. colonies of 0.1 mm or more in diameter were counted. The data was captured electronically using a validated software system (York Electronics). The plates were also examined for precipitates and microscopically, for microcolony growth.

Evaluation criteria:

A test was considered acceptable if, for each strain:
i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
ii) at least 2 of the vehicle control plates were within the following ranges:
TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.
iii) on at least 2 of the positive control plates there were at least x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100,
at least x 1.5 the mean vehicle control mutant numbers per plate.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli, at least a doubling of the mean concurrent vehicle control values at some
concentration of the test item. For S. typhimurium strain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, toxicity to the bacteria
generally, specific toxicity to the mutants and inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Statistics:

nda

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

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

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

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:

RANGE-FINDING/SCREENING STUDIES:
Toxicity to the bacteria was observed as a thinning of the background lawn of microcolonies at 500 μg per plate and upwards in both the presence and the absence of S9 mix. Precipitation of the test item occurred at the highest concentration, 5000 μg per plate, in both activation conditions.


COMPARISON WITH HISTORICAL CONTROL DATA:

Vehicle Control Groups:
The vehicle control values were within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium and E. coli.

Positive Control Groups:
The results obtained in the positive control groups were generally within the normal ranges expected for each bacterial strain and activation condition. The exception was E. coli in the second mutation assay, in the presence of S9 mix only. This was due to a dosing error with 2-AAN and the test was repeated.

4.2.4 Test Rejection
All tests were acceptable according to the study criteria except TA 100 in the first mutation assay, in the absence of S9 mix, which was rejected due to a dosing error at 333 μg per plate. In the second mutation assay, in the presence of S9 mix, TA 1537 was rejected due to a dosing error at 100 μg per plate. Also, E. coli was rejected due to a dosing error with 2-AAN. These parts of the test were repeated successfully.


ADDITIONAL INFORMATION:

Quality Control:
All strains were sensitive to crystal violet, whereas only the plasmid-containing strains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to u.v. light emitted over a period of 5-10 s from a lamp set at 254 nm. Increased sensitivity to u.v. light was demonstrated. These results are consistent with the known properties of these bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

WRS-2390TX did not induce mutagenic activity in any of the 5 bacterial strains used, in either activation condition.

Toxicity, observed as a thinning of the background lawn of microcolonies occurred in both mutation assays. In the first mutation assay, toxicity was observed at 333 μg per plate with all 4 Salmonella strains in the presence of S9 mix and with all 5 strains in the absence of S9 mix.

In the second mutation assay, toxicity was observed in the presence of S9 mix at 333 μg per plate with all 4 Salmonella strains. In the absence of S9 mix, toxicity was observed at 50 and 167 μg per plate with all 4 Salmonella strains and at 167 μg per plate with E. coli.

Toxicity was also observed in both mutation assays as a reduction in the number of revertant colonies. In the first mutation assay, in the presence of S9 mix, a reduction in colony numbers was observed with TA 98 and TA 100 at 333 μg per plate. In the absence of S9 mix, the presence of resistant colonies prevented the enumeration of revertant colonies of all 5 strains at 333 μg per plate.

In the second mutation assay, in the presence of S9 mix, a reduction in colony numbers was observed with TA 1535, TA 98 and TA 100 at 333 μg per plate. In the absence of S9 mix, a reduction in the number of revertant colonies was observed with TA 98 and E. coli at 167 μg per plate. The presence of resistant colonies prevented the enumeration of revertant colonies of TA 100 at 167 μg per plate.

In the second mutation assay retest, the presence of resistant colonies prevented the enumeration of revertant colonies of TA 1537 at 333 μg per plate.

No precipitation of the test item was observed in either mutation assay, in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that WRS-2390TX was not mutagenic to Salmonella typhimurium or Escherichia coli when tested in dimethylsulphoxide at concentrations extending into the toxic range.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability & repeatability).

Executive summary:

It was concluded that WRS-2390TX was not mutagenic to Salmonella typhimurium or Escherichia coli when tested in dimethylsulphoxide at concentrations extending into the toxic range.

Toxicity to the bacteria, observed as a thinning of the background lawn of microcolonies, occurred in both mutation assays. In the first assay (direct plate method), toxicity was observed at 333 μg per plate with all 4 Salmonella strains in the presence of S9 mix and with all 5 strains in the absence of S9 mix. In the second mutation assay (pre-incubation method), in the presence of S9 mix, toxicity was

observed at 333 μg per plate with all 4 Salmonella strains. In the absence of S9 mix, toxicity was observed at 50 and 167 μg per plate with all 4 Salmonella strains and at 167 μg per plate with E. coli.

Toxicity was also observed as a reduction in the number of revertant colonies. A reduction in colony numbers was observed with TA 98, TA 100 and TA 1535 (second assay only) at 333 μg per plate, in the presence of S9 mix. The presence of resistant colonies prevented the enumeration of revertant colonies of TA 1537 at 333 μg per plate.

In the absence of S9 mix, a reduction in colony numbers was observed with TA 98 and E. coli at 167 μg per plate. The presence of resistant colonies prevented the enumeration of revertant colonies of all 5 strains at 333 μg per plate and TA 100 at 167 μg per plate.

No precipitation of the test item was observed in either mutation assay in either the presence or the absence of S9 mix.

The methods used in this study conform to OECD Guideline No. 471, the European Commission Annex V Test Method B13 and B14, ICH Guidelines CPMP/ICH/141/95 and CPMP/ICH/174/95 and USA EPA 712-C-98-247. The ICH guidelines are also recognised by the Japanese Regulatory bodies. The study meets US EPA and FDA requirements and was conducted in accordance with Good Laboratory Practice