Carbamic acid,N,N'-[1,3-phenylenebis[methyleneiminocarbonylimino(methyl-3,1-phenylene)]]bis-di-C11-14-isoalkylesters, C13-rich

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(11-methyldodecyl) ((((1,3-phenylenebis(methylene)bis(azanediyl))bis(carbonyl) bis(azanediyl))bis(4-methyl-3,1-phenylene) dicarbamate

Cas Number:

865536-03-4

Molecular formula:

C52H80N6O6

IUPAC Name:

bis(11-methyldodecyl) ((((1,3-phenylenebis(methylene)bis(azanediyl))bis(carbonyl) bis(azanediyl))bis(4-methyl-3,1-phenylene) dicarbamate

Constituent 2

Reference substance name:

bis(11-methyldodecyl)(4-methyl-1,3-phenylene)dicarbamate

Molecular formula:

C35H62N2O4

IUPAC Name:

bis(11-methyldodecyl)(4-methyl-1,3-phenylene)dicarbamate

Constituent 3

Reference substance name:

11-methyldodecyl (3-(3-(3-(aminomethyl)benzyl)ureido)-4-methylphenyl)carbamate

Molecular formula:

C30H46N4O3

IUPAC Name:

11-methyldodecyl (3-(3-(3-(aminomethyl)benzyl)ureido)-4-methylphenyl)carbamate

Constituent 4

Reference substance name:

1,1'-(4-methyl-1,3-phenylene)bis(3-(3-(aminomethyl)benzyl)urea)

Molecular formula:

C25H30N6O2

IUPAC Name:

1,1'-(4-methyl-1,3-phenylene)bis(3-(3-(aminomethyl)benzyl)urea)

Test material form:

solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9, 2-Amino-anthracene, C14H11N; CAS-Nr.: 613-13-8

Details on test system and experimental conditions:

7 PERFORMANCE OF THE STUDY
7.1 Culture of Bacteria
12 hours before the start of each experiment, a stock culture of each strain was thawed
and an aliquot was placed into a culture flask containing 70 mL nutrient broth. The flasks
were incubated at 37°C for 12 hours.
7.2 Description of the Method
7.2.1 Preparations
In the days before each test, the media and solutions were prepared. Two days before the
test, the plates were sterilized and the first batches poured. On the day before the test, the
remaining plates were poured.
On the day of the test, the overnight cultures were checked for growth. The incubation
chamber was heated to 37 °C. The water bath was turned to 43 °C. The table surface was
disinfected.
The S9 mix was freshly prepared and stored at 0 °C.
7.2.3 Description of the Method
7.2.3.1 Plate incorporation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of
the test solution of the appropriate concentration were membrane filtrated into sterile vessels.
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-
solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the
water bath at 45 °C.
0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing
with 0.1 mL overnight culture of the respective strain and 0.5 mL phosphate buffer (only
for treatments without S9) or 0.5 mL S9 mix, 2 mL Top-Agar were added. The mixture
was gently vortexed, then poured on a minimal glucose plate and distributed evenly, using
a Drigalski spatula. The plates were closed, covered with brown paper and left to harden
for a few minutes, then inverted and placed in the dark incubator at 37 °C.
7.2.3.2 Pre-incubation method
Per strain and dose, four plates with and four plates without S9 mix were used. 10 mL of
the test solution of the appropriate concentration were membrane filtrated into sterile vessels.
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidinebiotin-
solution 0.5 mMol per 100 mL basis was added and the bottle was placed in the
water bath at 45 °C.
0.1 mL of the appropriate solution of the test item were given into a sterile tube. After mixing
with 0.1 mL overnight culture of the respective strain, 0.5 mL phosphate buffer (only
for treatments without S9) or 0.5 mL S9 mix were added. The mixture was incubated in an
incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated
through careful shaking. Then 2 mL top agar were added. The mixture was vortexed gently,
then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula.
The plates were closed, covered with brown paper and left to harden for a few minutes,
then inverted and placed in the dark incubator at 37 °C.

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction. A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

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 98

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 3

Key result

Species / strain:

S. typhimurium TA 100

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 4

Key result

Species / strain:

S. typhimurium TA 102

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 5

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

Any other information on results incl. tables

The test item didn’t show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item ADDUKT TI 65 - MXDA is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Applicant's summary and conclusion

Conclusions:

The test item is considered not mutagenic for the reasons given above. Also, the test item didn’t show any cytotoxicity towards the bacteria. The confirmation tests of the genotype didn’t show any irregularities. The control of the titre was above the demanded value. The positive controls didn’t evoke as many mutations as given by Prof. Ames. But the number of revertant colonies was in the range of the historical data of the laboratory (see page 37) and were definitely increased in comparison with the negative controls, as well as showing mutagenous potential of the diagnostic mutagenes. Some of the spontaneous revertants are lower than the values given by Prof. Ames; in comparison with the historical data of the LAUS GmbH, they were within the normal range. In Experiment 1, the strain TA 98 showed very high revertant values when compared with the historical data of the test facility. The revertants lay within the range which is given by Prof. Ames, though. The genotype confirmation for this strain did not show irregularities, therefore, this circumstance is not considered critical for the validity of the study outcome. For these reasons, the result of the test is considered valid.

Executive summary:

Two valid experiments were performed.

First Experiment:

Five concentrations of the test item, dissolved in dimethyl sulfoxid (DMSO) (ranging from

500 to 5 μg/plate) were used. Five genetically manipulated strains of Salmonella typhimurium

(TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both

in the presence and in the absence of a metabolic activation system (S9) for 48 hours,

using the plate incorporation method.

None of the concentrations caused an increase in the number of revertant colonies in the

tested strains. The test item didn’t show any mutagenic effects in the first experiment.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The

determined values for the spontaneous revertants of the negative controls were in the

normal range. All positive controls showed mutagenic effects with and without metabolic

activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using

three concentrations of the test item (ranging from 505 to 127 μg/plate) and a modification

in study performance (pre-incubation method).

The test item didn’t show mutagenic effects in the second experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The

determined values for the spontaneous revertants of the negative controls were in the

normal range. All positive controls showed mutagenic effects with and without metabolic

activation.

Under the conditions of the test, the test item didn’t show mutagenic effects towards Salmonella

typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

Therefore, no concentration-effect relationship could be determined.

The test item ADDUKT TI 65-MXDA is considered as

“not mutagenic under the conditions of the test”.