1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.02 - 03.03.2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EC and OECD guidelines and according to GLP principles.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

EC Number:

700-464-0

Cas Number:

99591-74-9

Molecular formula:

C2H4O6S2

IUPAC Name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Details on test material:

- Name of test material (as cited in study report): MMDS
- Substance type: organic
- Physical state: White crystal
- Analytical purity: 99.9%
- Purity test date: no data
- Lot/batch No.: 061201
- Expiration date of the lot/batch: no data
- Stability under test conditions: Stable at room temperature and dry condition
- Storage condition of test material: no data
- other: Degree of solubility in solvent: Water; Insoluble (hydrolysis), DMSO; 5wt% or more (Unstable), Acetone; 10wt% or more (Stable for 4 hours)

Method

Target gene:

histidine gene in Salmonella typhimurium
tryptophan gene in Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Dose-finding Assay: 1.2; 4.9; 20; 48; 313; 1250; 5000 μg/plate with and without S9 Mix
Main Assay: 20; 39; 78; 156; 313; 325; 1250; 2500 μg/plate without S9 Mix
20; 39; 78; 156; 313; 325; 1250; 2500; 5000 μg/plate with S9 Mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone 100% pure
- Justification for choice of solvent/vehicle: the test substance hydrolyses in water and unstable in DMSO. The preliminary test on solvent selection in the test facility revealed that the test substance was soluble at 10wt% in acetone and the solution was stable.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 0.01μg/plate for TA100

Remarks:

Without S9 Mix

Controls 2

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Remarks:

0.5 μg/plate for TA1535

Positive control substance:

sodium azide

Remarks:

Without S9 Mix

Controls 3

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 0.01 μg/plate for WP2 uvrA

Remarks:

Without S9 Mix

Controls 4

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide 0.1 μg/plate for TA98

Remarks:

Without S9 Mix

Controls 5

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine 1.0 μg/plate for TA1537

Remarks:

Without S9 Mix

Controls 6

Untreated negative controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 1.0 μg/plate for TA100; 2.0 μg/plate for TA1535 and TA1537; 0.5 μg/plate for TA98; 10 μg/plate for WP2 uvrA

Remarks:

With S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 10^9 cells/ml

NUMBER OF REPLICATIONS: duplicate at each dose level and in triplicate for negative control.

DETERMINATION OF CYTOTOXICITY: the growth inhibition of bacterial strain and the precipitation were examined, and the revertant colonies were counted

Evaluation criteria:

When the number of revertant colonies on the test substance treatment plate is increased 2-fold or more compared to the negative control value, and the increase is dose-dependent and reproducible between the dose-finding test and the main test, the test substance is judged to be positive for mutagenicity. Otherwise, the test substance is judged as negative. When the test substance is judged to be positive, the activity of mutagenicity is calculated.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The test substance increased the number of revertant colonies more than 2-fold compared to the negative control value and the increase was dose-dependent.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250μg/plate; extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The test substance increased the number of revertant colonies more than 2-fold compared to the negative control value and the increase was dose-dependent.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250μg/plate; extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250μg/plate; extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The test substance increased the number of revertant colonies more than 2-fold compared to the negative control value and the increase was dose-dependent

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250μg/plate; extremely toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250μg/plate; toxic

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Dose-finding assay: The test substance increased the number of revertant colonies more than 2-fold compared to the negative control value in TA100, TA1535 and TA98 under the conditions of S9 (±). On the other hand, the test substance did not increase the number of revertant colonies 2-fold or more compared to the negative control value in WP2 uvrA and TA1537 under the conditions of S9 (±).The growth inhibition of bacteria was observed at the doses and more described above.

Main test: the test substance increased the number of revertant colonies more than 2-fold compared to the negative control value in TA100, TA1535 and TA98 under the conditions of S9 (±), and the increase was dose-dependent. On the other hand, the test substance did not increase the number of revertant colonies 2-fold or more compared to the negative control value in WP2 uvrA and TA1537 under the conditions of S9 (±).
The precipitate of the test substance was not observed at any doses.

The value of maximum mutagenic activity was 1.30×103 rev./mg (at 313 μg/plate in TA100 with S9 in the dose-finding assay).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: MMDS tested at: 20, 39, 78, 156, 313, 625 and 1250 μg/plate in absence and presence of S9 mix

Any other information on results incl. tables

Table 1. Reults of Dose-finding Assay

Name of the testsubstance: MMDS
Testperiod: From February 8 to February 13, 2007
With(+) or Without(-) S9Mix	Dose (μg/plate)	Number of revertant coloniess/plate
		Base - pair substitution type			Frameshift type
		TA100	TA1535	WP2 uvrA	TA98	TA1537
S9Mix(-)	Negative control	128    97   97  ( 107)	16      9   22  ( 16)	30    37   37  ( 35)	32    29   23  ( 28)	10     4    9   (  8)
	1.2	110   99  ( 105)	11   16  ( 14)	38   39  ( 39)	27   22  ( 25)	6    6   (  6)
	4.9	113    98 ( 106)	11   20  ( 16)	29   40  ( 35)	22   31  ( 27)	4    1   (  3)
	20	101  107 ( 106)	23   19  ( 21)	20   35  ( 28)	27   30  ( 29)	1    4   (  3)
	78	161  147 ( 154)	26   16  ( 21)	40   29  ( 35)	45   39  ( 42)	8    5   (  7)
	313	292  204 ( 248)	56   40  ( 48)	45   60  ( 53)	66   75  ( 71)	7  10   (  9)
	1250	21*    18* ( 20)	42*   22* (  18)	37*   26* ( 32)	15    M  ( 15)	2*    5* (  4)
	5000	0N   0N  (  0)	0N    0N (   0)	18*     6* ( 12)	0N   0N  (  0)	0N    0N (  0)
S9Mix(+)	Negative control	150   149  137  ( 145)	22    12   17  ( 32)	49   46   44  ( 46)	35    32   44  ( 37)	11     7   16  ( 11)
	1.2	142  126  ( 134)	21   13  ( 17)	45   32  ( 39)	44   35  ( 40)	13   10  ( 12)
	4.9	145  130  ( 138)	14   20  ( 17)	34   36  ( 35)	42   44  ( 43)	11   10  ( 11)
	20	117  158  ( 138)	12   12  ( 12)	36   41  ( 39)	29   39  ( 34)	15   10  ( 13)
	78	242  199  ( 221)	23   40  ( 32)	55   54 (  55)	54   71  ( 63)	12   14  ( 13)
	313	544  560  ( 552)	47   35  ( 41)	72   53 (  63)	105    90 ( 98)	13   17  ( 15)
	1250	268  243  ( 256)	54   38  ( 46)	36*   43* ( 40)	29*    41* ( 35)	6*    4* (   5)
	5000	0N     0N (  0)	0N   0N ( 0)	21*    M* ( 21)	0N     0N (  0)	0N    0N (  0)
Positive control Without S9Mix	Name	AF－2	NaN3	AF－2	AF－2	lCR－191
	Dose(μg/plate)	0.01	0.5	0.01	0.1	1
	Number of coloniess/plate	536  543 ( 540)	577  505 ( 541)	116  119 ( 118)	493  459 ( 476)	3621  3532 (3577)
Positive Control With S9Mix	Name	2AA	2AA	2AA	2AA	2AA
	Dose(μg/plate)	1	2	10	0.5	2
	Number of coloniess/plate	1022  995 (1009)	389  440 ( 415)	1205  1322 (1264)	323  334 ( 329)	174  166 ( 170)

Notes:

* : Growth inhibition of bacteria

N : Bacteria growth was not observed

M : Small survival colonies were observed

Positive controls

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

NaN3: Sodium azide

ICR-191: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride

2AA: 2-Aminoanthracene

Table2. Results of Main study

Name of the testsubstance: MMDS
Testperiod： From February 15 to February 19, 2007
With(+) or Without(-) S9Mix	Dose (μg/plate)	Number of revertant coloniess/plate
		Base - pair substitution type			Frameshift type
		TA100	TA1535	WP2 uvrA	TA98	TA1537
S9Mix(-)	Negative control	105    92   88  ( 95)	6    10     5  (  7)	24   33   31 ( 29)	13   11   16 ( 13)	5    7    7 (  6)
	20	95  102 ( 99)	10   12  ( 11)	-  ( - )	8   18 ( 13)	-  ( - )
	39	121  103 ( 112)	8    9   (  9)	30   31 ( 31)	15   18 ( 17)	7    6 (  7)
	78	148  153 ( 151)	14   15  ( 15)	33   31 ( 32)	20   33 ( 27)	7    5 (  6)
	156	218  171 ( 195)	24   16  ( 20)	29   26 ( 28)	49   52 ( 51)	8    7 (  8)
	313	227  220 ( 224)	25   25  ( 25)	38   46 ( 42)	59   71 ( 65)	8   12 ( 10)
	625	220  251 ( 236)	40   30  ( 35)	37   55 ( 46)	65   68 ( 67)	13*    7* ( 10)
	1250	25*   38* ( 32)	30*  32* ( 31)	32*   38* ( 35)	15*   16* ( 16)	4*    3* (  4)
	2500	-  ( - )	-  ( - )	13*   20* ( 17)	-  ( - )	-  ( - )
S9Mix(+)	Negative  control	130 ( 135)  124 ( 130)	7      9    4   (  7)	37    34   37  ( 35)	23    36   29  ( 29)	13   10   11 ( 11)
	20	167  163 ( 165)	7    4   (  6)	45   32  ( 39)	36   39  ( 38)	-  ( - )
	39	163  183 ( 173)	11    7   (  9)	46   34 40	30   50 40	15   13 14
	78	225  199 ( 212)	17   18  ( 18)	41   45 43	51   40 46	10   16 13
	156	332  328 ( 330)	20   22  ( 21)	46   48 47	75  59 67	9   12 11
	313	486  496 ( 491)	34   47  ( 41)	59   61 60	70   65 68	8    9 9
	625	466  569 ( 518)	61   52  ( 57)	58   57 58	97   89 93	14   10 12
	1250	258*  283* ( 271)	41*   41* ( 41)	47*   25* ( 36)	43*   75* ( 59)	10*   11* ( 11)
	2500	-  ( - )	M*    M*	M*   13*   13	-  ( - )	1*    1* 1
	5000	-  ( - )	0N    0N ( 0)	-  ( - )	-  ( - )	-  ( - )
Positivecontrol Without S9Mix	Name	AF－2	NaN3	AF－2	AF－2	lCR－191
	Dose(μg/plate)	0.01	0.5	0.01	0.1	1
	Number of coloniess/plate	519  500 ( 510)	506  527 ( 517)	159  156 ( 158)	459  424 ( 442)	4471  4918 (4965)
Positive Control With S9Mix	Name	2AA	2AA	2AA	2AA	2AA
	Dose(μg/plate)	1	2	10	0.5	2
	Number of coloniess/plate	1074  1225 (1150)	423  432 ( 428)	1172  1182 (1177)	392  375 ( 384)	299  329 ( 314)

Notes:

* : Growth inhibition of bacteria

N : Bacteria growth was not observed

M : Small survival colonies were observed

Positive controls

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

NaN3: Sodium azide

ICR-191: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride

2AA: 2-Aminoanthracene

Table 3. Mutagenic activity

	Bacterial strain	-S9mix		＋S9mix
		Mutagenic activity	Doseused for calculation	Mutagenic activity	Doseused for calculation
Dose-finding assay	TA100	4.50×10^2	313	1.30×10^3	313
	TA1535	1.02×10^2	313	7.67×10	313
	WP2 uvrA	-	-	-	-
	TA98	1.37×10^2	313	1.95×10^2	313
	TA1537	-	-	-	-
Mainassay	TA100	6.41×10^2	156	1.28×10^3	156
	TA1535	1.03×10^2	78	1.41×10^2	78
	WP2 uvrA	-	-	-	-
	TA98	2.44×10^2	156	2.44×10^2	156
	TA1537	-	-	-	-

Unit: Mutagenic activity; rev./mg

Dose used for calculation [μg/plate]

: Maximum mutagenic activity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Based on the results, it was concluded that MMDS was positive for mutagenicity under the test conditions of this study.

Executive summary:

MMDS was evaluated for its mutagenic potential in the strains of Salmonella typhimurium, TA100, TA1535, TA98 and TA1537, and Escherichia coli WP2uvrA with or without metabolic activation.

In the dose-finding assay, the highest dose was set at 5000 μg/plate, and a total of 7 doses were set with a common ratio of 4.

In the dose-finding and main assays, the test substance increased the number of revertant colonies more than 2-fold compared to the negative control value in TA100, TA1535 and TA98 under the conditions of S9 (±), and the increase was dose-dependent. On the other hand, the test substance did not increase the number of revertant colonies 2-fold or more compared to the negative control value in WP2uvrA and TA1537 under the conditions of S9 (±).

The positive controls induced revertant colonies more than 2-fold of the negative control value in each strain. These results indicated the assays were performed properly.

Based on these results, it was concluded that MMDS was positive for mutagenicity under the test conditions of this study. The maximum mutagenic activity of the test substance was 1.30×10³rev./mg (at 313 μg/plate in TA100 with S9 in the dose-finding assay). In the dose-finding and main assays, the growth inhibition of bacteria by the test substance was observed at the doses.   The precipitate of the test substance was not observed at any doses.