[No public or meaningful name is available]

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:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid test guidelines, therefore it is considered to be relevant, adequate and reliable for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts

EC Number:

939-704-6

Cas Number:

867040-07-1

Molecular formula:

Molecular formula cannot be given as the substance is a mixture

IUPAC Name:

Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts

Test material form:

other: liquid

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Cytotoxicity test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate
Mutagenicity test: 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate (plate incorporation and preincubation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua ad iniectabilia

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide in aqua ad iniectabilia

Remarks:

(10 µg/plate):TA 1535, TA 100, without S9-mix

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-Nitro-fluorene in DMSO

Remarks:

(10 µg/plate):TA 98, without S9-mix

Controls 3

Positive controls:

yes

Positive control substance:

other: 9-Amino-acridine in ethanol, abs.

Remarks:

(100 µg/plate): TA 1537, without S9-mix

Controls 4

Positive controls:

yes

Positive control substance:

other: Mitomycin C in DMSO

Remarks:

( 10 µg/plate): TA 102, without S9-mix

Controls 5

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene in DMSO

Remarks:

( 10 µg/plate): TA 98, TA 102, TA 1537, with S9-mix

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene in DMSO

Remarks:

(2-4 µg/plate): TA 100, TA 1535, with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st independent experiment : in agar (plate incorporation);
2nd independent experiment : preincubation.

DURATION
1st independent experiment :
- Exposure duration: 48 h to 72 h
2nd independent experiment :
- Preincubation period: 20 min
- Exposure duration: 48 h to 72 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of revertant colonies, a clearing or diminution of the background lawn, or by the degree of survival of the treated cultures.
Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a sparse background lawn.

Evaluation criteria:

The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA 98, TA 100 and TA 102 and 3-fold of the vehicle control for TA 1535 and TA 1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed (The Spearman’s rank correlation coefficient) .
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

number of revertants compared with the vehicle control (p ≤ 0.05, U-test according to MANN and WHITNEY)
concentration-related increase of the revertants ((Spearman’s rank correlation coefficient)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: other: all strains tested in mutagenicity test, TA 100 without S9-mix in cytotoxicity test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimuriumstrains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.
The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.91 was used as the supplied test item contains only 34.40% active matter. Aqua ad iniectabilia was used as vehicle control.
The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µgtest item/plate in all test strains.
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to aconcentrationof 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.