Alkaline earth metal and zinc hydroxy(substituted)alkanoate and oxoalkanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-17 to 2018-07-31

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:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Target gene:

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
limit dose

Vehicle / solvent:

deionized water

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD, 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

For each strain and dose level, including the controls, three plates were used.

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspensio,
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 μg/plate

Evaluation criteria:

Data recording
The colonies were counted using a validated computer system (Major computerized systems), which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

Evaluation of results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

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

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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 4

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 5

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:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item occurred up to the highest investigated dose.

In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix. In experiment II reduced background growth was observed on the incubated agar plates from 2500 to 5000 μg/plate without S9 mix. The strains with S9 mix showed normal background growth.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with TP 1740 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Any other information on results incl. tables

Summary of Experiment I (Revertant colony counts, Mean +- SD)

Metabolic activation	Test Group	Dose level (per plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
without	deionised water		12 +- 2	14 +- 3	31 +- 4	189 +- 16	41 +- 4
	untreated		8 +- 3	13 +- 4	29 +- 4	189 +- 31	42 +- 6
	TP 1740	3 µg	11 +- 1	11 +- 1	29 +- 5	192 +- 13	34 +- 6
		10 µg	10 +- 3	11 +- 3	25 +- 5	183 +- 6	41 +- 7
		33 µg	9 +- 1	13 +- 2	24 +- 7	186 +- 20	44 +- 10
		100 µg	9 +- 4	13 +- 2	33 +- 5	192 +- 4	43 +- 9
		333 µg	8 +- 3	11 +- 1	31 +- 1	190 +- 7	45 +- 11
		1000 µg	10 +- 3	9 +- 2	36 +- 6	167 +- 7	45 +- 5
		2500 µg	10 +- 1	8 +- 2	23 +- 8	174 +- 12	39 +- 2
		5000 µg	12 +- 2	11 +- 2	25 +- 2	191 +- 13	41 +- 8
	NaN3	10 µg	1049 +- 99			1687 +- 59
	4 -NOPD	10 µg			376 +- 75
	4 -NOPD	50 µg		60 +- 9
	MMS	2.0 µL					808 +- 15
with	deionised water		15 +- 5	14 +- 4	38 +- 7	182 +- 14	43 +- 16
	untreated		15 +- 0	15 +- 5	47 +- 6	182 +- 15	50 +- 8
	TP 1740	3 µg	18 +- 4	15 +- 1	43 +- 3	172 +- 20	58 +- 11
		10 µg	16 +- 3	13 +- 3	44 +- 14	194 +- 6	60 +- 2
		33 µg	14 +- 4	17 +- 1	36 +- 4	167 +- 13	53 +- 12
		100 µg	16 +- 6	15 +- 4	38 +- 6	201 +- 8	40 +- 3
		333 µg	16 +- 5	16 +- 5	37 +- 0	166 +- 16	51 +- 11
		1000 µg	17 +- 4	19 +- 7	38 +- 10	173 +- 10	50 +- 11
		2500 µg	17 +- 6	21 +- 6	32 +- 2	170 +- 12	56 +- 2
		5000 µg	16 +- 6	16 +- 5	31 +- 5	190 +- 11	44 +- 4
	2 -AA	2.5 µg	381 +- 25	240 +- 24	3473 +- 387	3798 +- 99
	2 -AA	10.0 µg					234 +- 13

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed according to OECD guideline 471 and GLP to investigate the potential of TP 1740 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:                              33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred up to the highest investigated dose.

In experiment I the plates incubated with the test item showed normal background growth in all strains with and without S9 mix. In experiment II reduced background growth was observed on the incubated agar plates from 2500 to 5000 μg/plate without S9 mix. The strains with S9 mix showed normal background growth.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with TP 1740 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, TP 1740 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.