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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test available

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 2018 - 27 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test substance is completely miscible in water.
Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) from rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98): with and without S9: 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (all strains) with and without S9: 52, 164, 512, 1600 and 5000 μg/plate
Vehicle / solvent:
- Solvent used: Milli-Q water
- Justification for choice of solvent/vehicle: the solvent is according to OECD guideline 471 and the test substance formed a clear colorless solution in Milli-Q water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Remarks:
See table 1 for more details on postive control substances
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours
- Pre-incubation duration (only for experiment 2): 30 ± 2 minutes by 70 rpm

NUMBER OF REPLICATIONS: 3

PERFORMANCE OF THE ASSAY (Direct plate assay): Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C.
PERFORMANCE OF THE ASSAY (Pre-incubation assay): Before incubation, the following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water. After the pre-incubation period, the same approach as in the direct plate assay was used.

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Only in the pre-incubation assay, with S9-mix at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
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:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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
Additional information on results:
PRECIPITATION: In both experiments, no precipitation of the test item on the plates was observed at the start or at the end of the
incubation period.

CYTOTOXICITY
- Direct plate assay: no cytotoxicity observed at any of the tester strains at any of the tested concentrations.
- Pre-incubation assay: only observed in tester strain TA98 in the presence of S9-mix at the highest tested concentration. In all other
tester strains, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

MUTAGENICITY
- In both experiments: no increase in the number of revertants observed.

HISTORICAL CONTROL DATA
- The negative control values and the strain-specific positive control values were within the laboratory historical control data ranges.
Remarks on result:
other: Fluctuations in the number of revertant colonies below the laboratory historical control data range without a clear dose-relationship were observed but concluded not to be caused by toxicity of the test item.

Table 2 Historical Control Data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 – 27

3 – 20

3 – 23

8 - 41

8 – 55

61 – 176

60 - 160

10 – 59

9 - 67

Mean

10

11

6

6

16

22

110

106

26

33

SD

3

4

2

3

5

7

17

20

6

8

n

2458

2426

2402

2352

2416

2458

2473

2398

2237

2217

Table 3 Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

S9 -mix

-

+

-

+

-

+

Range

128 – 1530

73 – 1206

58 – 1407

54 – 1051

365 – 1978

250 – 1977

Mean

901

239

817

340

1355

903

SD

174

115

354

160

230

357

n

2400

2296

2051

2337

2357

2367

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1993

408 - 2379

93 – 1958

111 - 1359

Mean

905

1249

1059

444

SD

163

371

506

144

n

2402

2354

2153

2232

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2016 and May 2018. 

Conclusions:
Based on the results of an AMES test, performed according to OECD guideline 471 and GLP principles, Sodium salts of substituted amino acid (2) solution FC-C 13588 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification