Poly(substitutedmethyl)amino acid

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Remarks:

aquous solution

Details on test material:

Sodium salts of substituted amino acid (2) solution, FC-C 13588
Appearance: clear colorless solution

Specific details on test material used for the study:

The test substance is completely miscible in water.

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

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:

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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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. 

Applicant's summary and conclusion

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.