2,2-dimethylthiazolidine

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:

Experimental start date: 18 July 2017 Experimental completion date: 11 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

2,2-dimethylthiazolidine

EC Number:

242-980-4

EC Name:

2,2-dimethylthiazolidine

Cas Number:

19351-18-9

Molecular formula:

C5H11NS

IUPAC Name:

2,2-dimethylthiazolidine

Test material form:

liquid

Specific details on test material used for the study:

Physical state/Appearance: Liquid
Expiry Date: 21 September 2017
Storage Conditions: At room temperature

Method

Target gene:

histidine locus

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:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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 relevant 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

Vehicle / solvent:

DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA1535 and TA100

Positive control substance:

sodium azide

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA98, 50 µg/plate for TA1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Absence of S9-mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.0 µg/plate for TA102

Positive control substance:

other: methyl methane sulfonate

Remarks:

Absence of S9-mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate (10.0 µg/plate for TA102)

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Test Item Preparation
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was proven to be stable for this period based on Envigo Ref No. FC38CM.

Experimental Design and Study Conduct
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).

Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Experimental Performance
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 (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

Experiment II (Pre-Incubation)
In the pre-incubation assay 100 µL test solution (solvent or reference mutagen solution (positive control)), 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. 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.

Data Recording
The colonies were counted using a validated computer system (cf. 3.8, 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 (see tables of results). Due to extensive bacterial growth the colonies were partly counted manually.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding 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:

cytotoxicity

Remarks:

A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment I in strain TA 98 with S9 mix at 5000 µg/plate.

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:

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

Additional information on results:

This study was performed to investigate the potential of 2,2-Dimethylthiazolidin 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 TA102.
The assay was performed 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.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects other than the minor event mentioned in strain TA 98 occurred in the remaining test groups neither with nor without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,2-Dimethylthiazolidin 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. They showed a distinct in-crease in induced revertant colonies. In experiment I, the data in the solvent control of strain TA 102 with S9 mix were slightly above our historical control range. Since this deviation is rather small (653 colonies vs. 652 colonies), this effect is considered to be biologically irrelevant.

Analytical Control
To demonstrate that the test system was exposed to the intended concentrations of the test item in the mutagenicity test, the concentrations of the test item in solution were determined by GC. The analysis was performed with all concentrations, which were obtained by serial dilution of the highest concentration used. The values found by analysis were in agreement with the intended concentrations (recovery rate: 100 % ± 20 %), in general.
The test item concentrations of the samples 4 and 7 in experiment I (nominal concentration: 1.00 and 25 mg/mL, respectively) and samples 2 and 3 in experiment II (nominal concentrations: 1.00 and 3.33 mg/mL, respectively) deviated more than 20 % from the nominal concentrations of these samples. However, the values of the stock solutions were within the acceptance range of the indicated nominal value. Since all concentrations were obtained by serial dilution of the stock solution in DMSO, this deviation from the theoretical values to the measured values had no influence on the results of this study. Furthermore the deviations are considered as irrelevant since all of the affected concentrations are in the non-toxic concentration range.

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1810101	Study Code: Envigo 1810101
Experiment: 1810101 VVb	Date Plated: 18.07.2017
Assay Conditions:	Date Counted: 21.07.2017

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO		10 ± 1	8 ± 2	24 ± 2	153 ± 34	527 ± 4
	Untreated		11 ± 1	8 ± 2	22 ± 3	176 ± 4	564 ± 37
	2,2- Dimethyl-	3 µg	8 ± 3	8 ± 2	28 ± 7	157 ± 13	565 ± 31
	thiazolidin	10 µg	8 ± 2	8 ± 2	21 ± 7	159 ± 16	554 ± 41
		33 µg	12 ± 4	9 ± 3	24 ± 3	154 ± 11	527 ± 11
		100 µg	7 ± 3	10 ± 2	24 ± 3	145 ± 9	554 ± 7
		333 µg	8 ± 3	9 ± 2	26 ± 4	164 ± 19	581 ± 50
		1000 µg	8 ± 2	11 ± 5	25 ± 4	146 ± 6	549 ± 20
		2500 µg	8 ± 3	6 ± 3	29 ± 5	150 ± 4	480 ± 31
		5000 µg	7 ± 2	7 ± 2	23 ± 4	152 ± 13	448 ± 10
	NaN3	10 µg	1111 ± 102			1743 ± 37
	4-NOPD	10 µg			276 ± 17
	4-NOPD	50 µg		87 ± 4
	MMS	2.0 µL					3618 ± 192
With Activation	DMSO		9 ± 2	12 ± 4	47 ± 2	141 ± 6	653 ± 13
	Untreated		10 ± 5	10 ± 1	33 ± 14	156 ± 21	675 ± 7
	2,2- Dimethyl-	3 µg	9 ± 2	13 ± 5	36 ± 11	127 ± 6	668 ± 31
	thiazolidin	10 µg	6 ± 2	11 ± 5	36 ± 6	139 ± 18	641 ± 36
		33 µg	9 ± 2	12 ± 3	33 ± 9	129 ± 10	597 ± 24
		100 µg	13 ± 6	13 ± 3	26 ± 2	118 ± 3	598 ± 42
		333 µg	10 ± 3	10 ± 3	37 ± 8	112 ± 16	683 ± 28
		1000 µg	8 ± 3	15 ± 1	29 ± 2	147 ± 8	589 ± 20
		2500 µg	9 ± 3	9 ± 3	25 ± 9	133 ± 20	509 ± 23
		5000 µg	8 ± 2	6 ± 1	21 ± 5	136 ± 13	447 ± 38
	2-AA	2.5 µg	384 ± 61	109 ± 6	3775 ± 45	2194 ± 559
	2-AA	10.0 µg					1637 ± 110

Key to Positive Controls
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine

Summary of Experiment II

Study Name: 1810101	Study Code: Envigo 1810101
Experiment: 1810101 HV2 Pre	Date Plated: 05.09.2017
Assay Conditions:	Date Counted: 11.09.2017

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO		12 ± 5	10 ± 4	26 ± 6	127 ± 7	479 ± 17
	Untreated		11 ± 2	9 ± 1	34 ± 3	173 ± 27	501 ± 35
	2,2- Dimethyl-	33 µg	11 ± 1	7 ± 2	30 ± 3	118 ± 4	491 ± 8
	thiazolidin	100 µg	8 ± 3	8 ± 2	29 ± 4	120 ± 19	460 ± 20
		333 µg	10 ± 3	11 ± 0	29 ± 1	123 ± 10	437 ± 25
		1000 µg	11 ± 2	10 ± 1	26 ± 1	118 ± 16	431 ± 25
		2500 µg	12 ± 3	7 ± 3	25 ± 3	111 ± 5	434 ± 47
		5000 µg	13 ± 2	10 ± 1	21 ± 5	101 ± 6	482 ± 65
	NaN3	10 µg	1080 ± 49			2002 ± 95
	4-NOPD	10 µg			1444 ± 24
	4-NOPD	50 µg		86 ± 4
	MMS	2.0 µL					4154 ± 182
With Activation	DMSO		11 ± 4	12 ± 2	32 ± 6	131 ± 10	642 ± 31
	Untreated		16 ± 1	13 ± 3	43 ± 10	183 ± 16	648 ± 4
	2,2- Dimethyl-	33 µg	10 ± 4	12 ± 3	38 ± 11	145 ± 8	634 ± 24
	thiazolidin	100 µg	12 ± 2	13 ± 3	42 ± 11	134 ± 17	622 ± 24
		333 µg	9 ± 3	16 ± 4	44 ± 7	114 ± 14	594 ± 4
		1000 µg	13 ± 3	16 ± 6	30 ± 9	134 ± 13	556 ± 45
		2500 µg	13 ± 5	6 ± 1	35 ± 6	121 ± 9	462 ± 35
		5000 µg	13 ± 5	6 ± 2	33 ± 10	106 ± 9	483 ± 25
	2-AA	2.5 µg	386 ± 8	91 ± 17	3724 ± 338	2931 ± 207
	2-AA	10.0 µg					1910 ± 84

Key to Positive Controls
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine

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:

 Summary

This study was performed to investigate the potential of 2,2-Dimethylthiazolidin 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 TA102.

The assay was performed 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.

 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in experiment I in strain TA 98 with S9 mix at 5000 µg/plate. No other toxic effects occurred in the remaining test groups neither with nor without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,2-Dimethylthiazolidin 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.

 

Conclusion

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, 2,2-Dimethylthiazolidin is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.