1,3,2-dioxathiolane 2,2-dioxide

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:

Study initiation: 22 November 2010, Final report: 22 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Korea

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3,2λ⁶-dioxathiolane-2,2-dione

EC Number:

600-809-4

Cas Number:

1072-53-3

Molecular formula:

C2H4O4S

IUPAC Name:

1,3,2λ⁶-dioxathiolane-2,2-dione

Test material form:

solid

Specific details on test material used for the study:

Chemical name : 1,3,2-Dioxathiolane, 2,2-dioxide
Product name: ESA
Lot No. : Not available
Received date : 18 November, 2010
Appearance : Lemon yellow solid
Purity : 99.1 %
Stability : Stable in recommended storage condition

Method

Target gene:

histidine and tryptophan

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:

Liver of Sprague-Dawley male rat induced by Aroclor-1254, 500 mg/kg i.p.

Test concentrations with justification for top dose:

•In the absence of metabolic activation system (S9 mix(-))
TA100, TA1537, WP2uvrA
0, 5, 10, 20, 39, 78, 156, 313 µg/plate
TA1535
0, 39, 78, 156, 313, 625, 1250, 2500 µg/plate
TA98
0, 0.6, 1.25, 2.5, 5, 10, 20, 39 µg/plate
•In the presence of metabolic activation system (S9 mix(+))
0, 5, 10, 20, 39, 78, 156, 313 µg/plate

Vehicle / solvent:

The test substance showed high solubility in DMSO but crystals were formed after. DMSO was not suitable for use in the test. The test substance showed high solubility in 1,4-Dioxane and did not react with the 1,4-Dioxane. Therefore, 1,4-Dioxane was selected to negative control.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1,4-Dioxane

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1,4-Dioxane

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for strain TA1537

Positive control substance:

other: 9-Aminoacridine hydrochloride hydrate

Remarks:

Without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1,4-Dioxane

True negative controls:

no

Positive controls:

yes

Remarks:

0.01 µg/plate for strains TA100 and WP2uvrA, 0.1 µg/plate for strain TA98

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1,4-Dioxane

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for strain TA98, 1.0 µg/plate for strain TA100, 2.0 µg/plate for strains TA1535 and TA1537, 10 µg/plate for strain WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Details on test system and experimental conditions:

Condition of pre-cultivation
Nutrient broth: No. 2, Oxoid, 588285
Cultivation time: 10 hours (Stop incubation in the early stationary phase)
Shaking incubator: Model: SHKE-5000-1CE
Incubation method: Shaking type: rotation (RPM: 180 /min) Rotation diameter: 2.54 cm
Culture vessel: Erlenmeyer flask Capacity: 50 ml
Volume of culture medium: 15 ml
Volume of inoculum: 30 ml

Preparation of test substance
The test substance was dissolved in 1,4-dioxane and then, serially diluted (two-fold) from highest concentration.

Method
This study was conducted using preincubation method with and without metabolic activation system (S9).

Test condition
Preincubation: 37 ºC, 20 min
Incubation: 37 ºC, 48 hours

Experiments with test item and controls were performed in triplicate

Method of observation, measurement and analysis

Colony counting
The number of revertants were counted only inside area of plate (diameter : 90 mm) by manual measurement using electronic register (Model 570, SUNTEX, Taiwan).
Test method of growth inhibition
All plate was observed with the naked eye.

Measurement of bacterial concentration
It was performed by step-dilution method according to the Standard Operation Procedure, KCL.

Sterility test
0.1 ml of S9 mix and test substance were mixed with top agar respectively, and then, it was added onto minimum glucose agar plate. The plate was incubated at 37ºC for 48 hours. Since the bacteria did not grow after incubation, it was confirmed that the test was performed aseptically.

Evaluation criteria:

The test result was recorded experimental value, average value and standard deviation for the number of revertant colonies per plate. The result was judged as ‘Positive’, if there is a dose-dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Also, the number of revertant colony should increase more than 2 times than the negative control group.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Increase in the number of revertant colonies was seen in TA100, TA1535 and WP2uvrA strains compared with negative control groups in the absence of metabolic activation system. Increase in the number of revertant colonies was seen in TA100, TA1535, WP2uvrA and TA1537 strains compared with negative control groups in the presence of metabolic activation system.

Preliminary range-finding test was performed to determine dose levels at the main test.
Preliminary range-finding test was carried out on 313, 625, 1250, 2500, 5000 µg/plate (two-fold serial dilutions). As a result of preliminary range-finding test, cytotoxicity was observed in TA98, TA100, TA1537 and WP2uvrA strains more than 313 µg/plate dose levels regardless metabolic activation system. In the case of TA1535 strain, cytotoxicity was observed at a concentration of 2500 µg/plate or higher in the absence of metabolic activation system, and cytotoxicity was observed at a concentration of 313 µg/plate or higher in the presence of metabolic activation system. In the case of TA98 strain, main test (II) was performed on 0~39 µg/plate because cytotoxicity was observed at TA98 strain more than 39 µg/plate dose levels in absence of metabolic activation system.

As a result of main test, cytotoxicity was observed in TA100, TA1537 strains more than 156 µg/plate dose levels at absence of metabolic activation system. At the concentration of 2500 µg/plate for TA1535, 313 µg/plate for WP2uvrA and 39 µg/plate and above for TA98, cytotoxicity was observed in the absence of metabolic activation system. At the concentration of 156 µg/plate and above for TA100, 313 µg/plate for TA98, TA1535, TA1537 and WP2uvrA, cytotoxicity was observed in the presence of metabolic activation system.
When compared to the negative control, significant increases in the number of revertant colonies were observed in TA100, TA1535 and WP2uvrA strains regardless presence of metabolic activation system. In the case of TA1537 strain, significant increases in the number of revertant colonies were observed in presence of metabolic activation system.

Sterility of the solvent and S9 mix were certified by the sterility test. It was counted that the number of revertant colonies of positive control groups and negative control groups were within or close to the range of the historical data. Therefore, this test was performed properly.

As the results, ESA did induce reverse mutation in this test condition.

Any other information on results incl. tables

Test results (Main study)

Metabolic activation		Dose (µg/plate)	Number of colony/plate
			Base-pair substitution type						Frameshift type
			TA100	Dose (µg/plate)	TA1535	Dose (µg/plate)	WP2uvrA	Dose (µg/plate)	TA98	Dose (µg/plate)	TA1537
S9 Mix (-)		0	70 64 61	0	9 6 4	0	45 43 46	0	13 13 15	0	3 6 5
		Mean ± SD	66 ± 4.6	Mean ± SD	6 ± 2.5	Mean ± SD	45 ± 1.5	Mean ± SD	14 ± 1.2	Mean ± SD	5 ± 1.5
		5	107 138 89	39	315 286 272	5	750 636 688	5	11 14 5	5	3 8 7
		Mean ± SD	111 ± 24.8	Mean ± SD	291 ± 21.9	Mean ± SD	691 ± 57.1	Mean ± SD	10 ± 4.6	Mean ± SD	6 ± 2.6
		10	73 76 109	78	418 456 350	10	854 938 1104	10	6 14 9	10	5 5 3
		Mean ± SD	86 ± 20.0	Mean ± SD	408 ± 53.7	Mean ± SD	965 ± 127.2	Mean ± SD	10 ± 4.0	Mean ± SD	4 ± 1.2
		20	144 126 150	156	764 794 752	20	1824 1680 1542	20*	8 13 7	20	3 5 2
		Mean ± SD	140 ± 12.5	Mean ± SD	770 ± 21.6	Mean ± SD	1682 ± 141.0	Mean ± SD	9 ± 3.2	Mean ± SD	3 ± 1.5
		39	175 200 232	313	924 996 984	39	2200 1836 2068	39*	P/C P/C P/C	39	8 2 7
		Mean ± SD	202 ± 28.6	Mean ± SD	968 ± 38.6	Mean ± SD	2035 ±184.3	Mean ± SD	- ± -	Mean ± SD	6 ± 3.2
		78	150 107 134	625	870 1176 1300	78	2306 2280 2784	78*	P/C P/C P/C	78	5 6 4
		Mean ± SD	130 ± 21.7	Mean ± SD	1115 ± 221.3	Mean ± SD	2457 ± 283.8	Mean ± SD	- ± -	Mean ± SD	5 ± 1.0
		156*	P/C P/C P/C	1250	788 1332 996	156	2894 2502 2778	156*	P/C P/C P/C	156*	P/C P/C P/C
		Mean ± SD	- ± -	Mean ± SD	1039 ± 274.5	Mean ± SD	2725 ± 201.4	Mean ± SD	- ± -	Mean ± SD	- ± -
		313*	P/C P/C P/C	2500*	0 0 0	313*	P/C P/C P/C	313*	0 0 0	313*	P/C P/C P/C
		Mean ± SD	- ± -	Mean ± SD	0 ± 0	Mean ± SD	- ± -	Mean ± SD	0 ± 0	Mean ± SD	- ± -
S9 Mix (+)		0	100 124 137	0	8 7 8	0	75 85 70	0	27 22 38	0	4 7 3
		Mean ± SD	120 ± 18.8	Mean ± SD	8 ± 0.6	Mean ± SD	77 ± 7.6	Mean ± SD	29 ± 8.2	Mean ± SD	5 ± 2.1
		5	324 354 370	5	812 930 960	5	326 327 328	5	26 33 38	5	5 7 7
		Mean ± SD	349 ± 23.4	Mean ± SD	901 ± 78.2	Mean ± SD	327 ± 1.0	Mean ± SD	32 ± 6.0	Mean ± SD	6 ± 1.2
		10	620 498 712	10	1106 1118 1110	10	1308 1340 1284	10	37 41 45	10	3 6 3
		Mean ± SD	610 ± 107.3	Mean ± SD	1111 ± 6.1	Mean ± SD	1311 ± 28.1	Mean ± SD	41 ± 4.0	Mean ± SD	4 ± 1.7
		20	888 734 688	20	1792 1728 1420	20	1960 2272 2040	20	48 61 71	20	7 4 7
		Mean ± SD	770 ± 104.7	Mean ± SD	1647 ± 198.9	Mean ± SD	2091 ± 162.1	Mean ± SD	60 ± 11.5	Mean ± SD	6 ± 1.7
		39	852 860 854	39	2104 2000 1740	39	3044 3624 3048	39	45 58 62	39	15 15 10
		Mean ± SD	855 ± 4.2	Mean ± SD	1948 ±187.5	Mean ± SD	3238 ± 333.7	Mean ± SD	55 ± 8.9	Mean ± SD	13 ± 2.9
		78	407 329 443	78	1680 1792 1816	78	2576 3112 2880	78	43 56 47	78	18 13 20
		Mean ± SD	393 ± 58.3	Mean ± SD	1763 ± 72.6	Mean ± SD	2856 ± 268.8	Mean ± SD	49 ± 6.7	Mean ± SD	17 ± 3.6
		156*	P/C P/C P/C	156	644 1116 1324	156	3144 2884 3196	156*	5 5 7	156	5 3 6
		Mean ± SD	- ± -	Mean ± SD	1028 ± 348.4	Mean ± SD	3075 ±167.2	Mean ± SD	6 ± 1.2	Mean ± SD	5 ± 1.5
		313*	P/C P/C P/C	313*	P/C P/C P/C	313*	P/C P/C P/C	313*	P/C P/C P/C	313*	0 0 0
		Mean ± SD	- ± -	Mean ± SD	- ± -	Mean ± SD	- ± -	Mean ± SD	- ± -	Mean ± SD	0 ± 0
Positive controls	S9 Mix (-)	Positive controls	AF-2	Positive controls	NaN3	Positive controls	AF-2	Positive controls	AF-2	Positive controls	9-AA
		Dose (µg/plate)	0.01	Dose (µg/plate)	0.5	Dose (µg/plate)	0.01	Dose (µg/plate)	0.1	Dose (µg/plate)	80
		Number of colony	499 401 410	Number of colony	237 198 225	Number of colony	280 228 223	Number of colony	501 345 396	Number of colony	2464 2260 2584
		Mean ± SD	437 ± 54.2	Mean ± SD	220 ±20.0	Mean ± SD	244 ± 31.6	Mean ± SD	414 ± 79.5	Mean ± SD	2436 ± 163.8
	S9 Mix (+)	Positive controls	2-AA	Positive controls	2-AA	Positive controls	2-AA	Positive controls	2-AA	Positive controls	2-AA
		Dose (µg/plate)	1	Dose (µg/plate)	2	Dose (µg/plate)	10	Dose (µg/plate)	1	Dose (µg/plate)	2
		Number of colony	448 592 626	Number of colony	207 201 251	Number of colony	280 261 264	Number of colony	246 234 259	Number of colony	191 155 167
		Mean ± SD	555 ± 94.5	Mean ± SD	220 ± 27.3	Mean ± SD	268 ± 10.2	Mean ± SD	246 ± 12.5	Mean ± SD	171 ± 18.3

* cytotoxicity

Test results (Main study Ⅱ)

Metabolic activation		Dose (µg/plate)
			Number of colony/plate
			Base-pair substitution type			Frameshift type
			TA100	TA1535	WP2uvrA	TA98	TA1537
S9 Mix (-)		0				28 22 24
		Mean ± SD				25 ± 3.1
		0.6				26 27 24
		Mean ± SD				26 ± 1.5
		1.25				23 29 22
		Mean ± SD				25 ± 3.8
		2.5				27 24 30
		Mean ± SD				27 ± 3.0
		5				28 28 34
		Mean ± SD				30 ± 3.5
		10				30 33 33
		Mean ± SD				32 ± 1.7
		20				21 25 30
		Mean ± SD				25 ± 4.5
		39*				08 9 8
		Mean ± SD				8 ± 0.6
S9 Mix (+)		0				24 27 21
		Mean ± SD				24 ± 3.0
		5				28 27 37
		Mean ± SD				31 ± 5.5
		10				25 32 33
		Mean ± SD				30 ± 4.4
		20				40 43 33
		Mean ± SD				36 ± 5.1
		39				45 37 46
		Mean ± SD				43 ± 4.9
		78				46 36 57
		Mean ± SD				46 ± 10.5
		156				30 22 20
		Mean ± SD				24 ± 5.3
		313*				P/C P/C P/C
		Mean ± SD				- ± -
Positive controls	S9 Mix (-)	Positive controls	AF-2	NaN3	AF-2	AF-2	9-AA
		Dose (µg/plate)	0.01	0.5	0.01	0.1	80
		Number of colony				490 466 452
		Mean ± SD				469 ± 19.2
	S9 Mix (+)	Positive controls	2-AA	2-AA	2-AA	2-AA	2-AA
		Dose (µg/plate)	1	2	10	1	2
		Number of colony				564 540 572
		Mean ± SD				559 ± 16.7

* cytotoxicity

Applicant's summary and conclusion

Conclusions:

When compared to the negative control, significant increases in the number of revertant colonies were observed in TA100, TA1535 and WP2uvrA strains regardless of presence of metabolic activation system. In the case of TA1537 strain, significant increases in the number of revertant colonies were observed in presence of metabolic activation system.
ESA did induce reverse mutation in under the conditions of this test.

Executive summary:

Summary

To evaluate mutagenic potential of ESA in bacteria, bacterial reverse mutation study was performed with preincubation method using four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the presence and absence of metabolic activation system (S9 mix). The test substance was dissolved 1,4-dioxane and then it was applied to the test strains at the dose levels of followings.

•In the absence of metabolic activation system (S9 mix(-))

TA100, TA1537, WP2uvrA: 0, 5, 10, 20, 39, 78, 156, 313 ㎍/plate

TA1535: 0, 39, 78, 156, 313, 625, 1250, 2500 ㎍/plate

TA98: 0, 0.6, 1.25, 2.5, 5, 10, 20, 39 ㎍/plate

•In the presence of metabolic activation system (S9 mix(+))

0, 5, 10, 20, 39, 78, 156, 313 ㎍/plate

Increase in the number of revertant colonies was seen in TA100, TA1535 and WP2uvrA strains compared with negative control groups in the absence of metabolic activation system. Increase in the number of revertant colonies was seen in TA100, TA1535, WP2uvrA and TA1537 strains compared with negative control groups in the presence of metabolic activation system.

Considering these findings, it is concluded that the test substance ESA does induce reverse mutation in TA100, TA1535, WP2uvrA and TA1537 strains which were used in this study.