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Toxicological information

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:
November 22, 2012 to December 3, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
“OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test” (Adopted: July 21, 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
“Bacterial Reverse Mutation Test” of “Mutagenicity Test” stipulated in the “Test Methods of the New Chemical Substances etc.” (March 31, 2011, Yakushokuhatsu 0331 No. 7, Heisei 23.03.29 Seikyoku No. 5, Kanpokihatsu No. 110331009, partly amended by Yakushokuhatsu 0402 No. 1, Heisei 24.03.28 Seikyoku No. 2, Kanpokihatsu No. 120402001 on April 2, 2012)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
“Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” (Public Notice No. 77, the Ministry of Labour, September 1, 1988, amended by Public Notice No. 67, the Ministry of Labour, June 2, 1997 and Public Notice No. 120, the Ministry of Labour, December 25, 2000) and “Specific Test Methods and Test Result Evaluation Methods for Microbial Mutagenicity Test” (Memorandum, Manager of Chemical Substances Investigation Division, Industrial Safety and Health Department, Labour Standards Bureau, the Ministry of Labour, February 8, 1999)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one
EC Number:
835-272-7
Cas Number:
256374-76-2
Molecular formula:
C12H26N2O6P2
IUPAC Name:
2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one
Test material form:
solid: particulate/powder
Details on test material:
Name
Name: Reaction products of ethane-1,2-diamine, phosphoryl=trichloride and 2,2-dimethylpropane-1,3-diol which makes N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane=2-oxide-2-yl)ethane-1,2-diamine as a main component
Other name: SH-0850
CAS number: 256374-76-2 (main component)

Structural formula
Molecular formula: C12H26N2O6P2 (main component)
Molecular weight: 356.29 (main component)

Provided sample
Purity of the test substance: 100%
Lot number: SK-241002

Physical-chemical properties
Solubility in water: Less than 0.03% (w/w) by visual observation
Melting point: 277 °C
Appearance at normal temperatures: White powder

Storage condition
The test substance was stored in a dark place at room temperature.

Precaution for handling
Protective gloves, mask, glasses and clothes were put on in order to avoid contacts with skin or eyes and inhalation.
Specific details on test material used for the study:
No further details specified in the study report.

Method

Target gene:
histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Species and reason for selection
Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 were used as the tester strains in this study. Salmonella typhimurium strains were generously provided by Dr. Taijiro Matsushima, Japan Bioassay Research Center on March 13, 2003 and September 20, 2003, respectively. The use of these tester strains in the microbial mutagenicity test and bacterial reverse mutation test is recommended in “Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” and “Test Methods of the New Chemical Substances etc.”, and “OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test”.
Storage
The tester strains were preliminarily checked for the amino acid requirement, ultraviolet sensitivity, rfa wall mutation, presence or absence of plasmid pKM101 and for the negative and positive control value. It was confirmed that these tester strains had the appropriate properties.
Dimethyl sulfoxide was mixed into the culture medium of the tester strains at a volume ratio of 10:0.9, and the mixtures were dispensed and stored as working stocks at -80 °C or below in an ultra-deep freezer 3 in Ames test room 1. The working stocks were freshly thawed before use.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Species and reason for selection
Escherichia coli strain WP2uvrA was used as the tester strain in this study. Escherichia coli strain was generously provided by Dr. Taijiro Matsushima, Japan Bioassay Research Center on March 13, 2003 and September 20, 2003, respectively. The use of the tester strain in the microbial mutagenicity test and bacterial reverse mutation test is recommended in “Public Notice stipulating the Standards determined by the Minister of Health, Labour and Welfare based on the provisions in Paragraph 1, Article 57-2 (current: Paragraph 1, Article 57-3) of the Industrial Safety and Health Law” and “Test Methods of the New Chemical Substances etc.”, and “OECD Guidelines for the Testing of Chemicals, No. 471, Bacterial Reverse Mutation Test”.
Storage
The tester strain was preliminarily checked for the amino acid requirement, ultraviolet sensitivity, rfa wall mutation, presence or absence of plasmid pKM101 and for the negative and positive control value. It was confirmed that the tester strain had the appropriate properties. Dimethyl sulfoxide was mixed into the culture medium of the tester strains at a volume ratio of 10:0.9, and the mixtures were dispensed and stored as working stocks at -80 °C or below in an ultra-deep freezer 3 in Ames test room 1. The working stocks were freshly thawed before use.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
1) Rat liver S9
S9 (lot number 12090706, manufactured on September 7, 2012, Oriental Yeast Co., Ltd.,) prepared from the livers of 7-week old male SD rats (body weight: 208.5 ± 8.8 g) treated (intraperitoneal administration) with the combination of phenobarbital (single dose of 30 mg/kg and three doses of 60 mg/kg) and 5,6-benzoflavone (single dose of 80 mg/kg) was used. S9 was stored at -80 °C or below frozen in an ultra-deep freezer 3 in Ames test room 1 until use. The frozen S9 was freshly thawed just before use.

2) Composition of S9 mix
S9 mix was freshly prepared before use in the test. 1 mL of S9 mix contained 8 μM of MgCl2, 33 μM of KCl, 5 μM of glucose-6-phosphate, 4 μM of NADPH, 4 μM of NADH, 100 μM of sodium phosphate buffer (pH 7.4) and 0.1 mL of S9.
Test concentrations with justification for top dose:
Based on the result of range-finding test, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313μg/plate in the absence of S9 mix of any tester strains for Main test-1. In the presence of S9 mix, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313μg/plate of any tester strains for Main test-1.

Main test-2
As the result of Main test-1, no increase to 2-fold or more over the negative control value in the number of revertant colonies was noted for all tester strains in the presence and absence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the presence and absence of S9 mix. Therefore, 5 dose levels by 2-fold serial dilutions were set from 5000 to 2500, 1250, 625 and 313 μg/plate of any tester strains for Main test-2.
Vehicle / solvent:
Negative control substance (vehicle)
Name: DMSO
Manufacturer, lot number, purity and grade
Manufacturer: Dojindo Laboratories
Lot number: DB136
Purity: 99.9 %
Grade: Pure solvent for ultraviolet absorption spectrum

Reason for vehicle selection
The test substance was not soluble in distilled water (50.0 mg/mL) and acetone (100 mg/mL) but suspended in DMSO (50.0 mg/mL). Heat generation, bubbling and change of color tone were not observed for 50.0 mg/mL suspension using DMSO up to 2 hours after preparation at room temperature. Therefore, it was determined to be stable and DMSO was selected as vehicle.

Storage conditions
The vehicle was frozen (allowable range: -30 - -10 oC) and stored in Freezer 5 in Ames experiment Room 1.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracence (2AA); ICR-191
Details on test system and experimental conditions:
Experimental method
The tests were performed by the pre-incubation method in the absence and presence of S9 mix. Three plates were used for the negative control and 2 plates per dose level were used for each positive control and the test substance treatment groups in the dose-range finding test. In the main studies 3 plates per dose level were used for each negative control, positive control and test substance treatment groups. The study code number, the name of bacterial strain, presence or absence of S9 and the dose level were written on each plate for identification.

Operational procedure
In each operation, a mixture consisting of 0.1 mL of the test substance solution, the solvent or the positive control solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix and 0.1 mL of bacterial culture solution were added in a test tube and shook at 37 ± 0.5 °C for 20 minutes. 2 mL of the soft agar was then added to the test tube and the mixture was overlaid onto the surface of the plate. After the plate was incubated at 37 ± 0.5 °C for 48 hours, the number of revertant colonies formed on the plate was counted.

Sterility test
After 2 mL of soft agar was added to each of the test substance solution of the highest dose level (0.1 mL) and S9 mix (0.5 mL), these mixtures were overlaid onto the surface of the plates. The plates were incubated at 37 ± 0.5 °C for 48 hours and the presence or absence of contamination was determined for each plate.

Observation and counting
Observation
At the end of culture period, the presence or absence of precipitation of the test substance was determined by visual examination and the presence or absence of growth inhibition was determined using a stereoscopic microscope.

Counting
The number of colonies was counted using a colony analyzer (CA-11D, System Science Co., Ltd.,) for all plates. When using the colony analyzer, the colony count was corrected for the area and count loss and used as the number of revertant colony.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
The test result was considered to be positive when the number of revertant colonies was increased to 2-fold or more over the negative control value and the mode of increase was dose dependent or reproducible, and all other test results were considered to be negative.
Statistics:
No statistical procedure was applied.

Results and discussion

Test resultsopen allclose all
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
True negative controls validity:
not applicable
Positive controls validity:
valid
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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
True negative controls validity:
not applicable
Positive controls validity:
valid
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose-range finding test
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for TA100 in the absence of S9 mix. No growth inhibition was observed. Precipitation of the test substance was not observed.

Main test-1
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for all tester strains in the absence and presence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the absence and presence of S9 mix.

Main test-2
There was no increase to 2-fold or more over the negative control value in the number of revertant colonies for all tester strains in the absence and presence of S9 mix. No growth inhibition was observed for any test conditions. Precipitation of the test substance was not observed in the absence and presence of S9 mix.

Discussion
As the result of the tests, the number of revertant colonies was below 2-fold over the negative control value for all tester strains in the absence and presence of S9 mix. The mutagenicity of the test substance was judged negative.
The number of revertant colonies for positive control was 2-fold or more value over the negative control, and the numbers of revertant colonies for the negative control and positive controls were within the range of the historical data. Furthermore, the test system was found to be free of contamination. Therefore, it was judged that the study was performed properly.

Any other information on results incl. tables

Table of the test result (Main test-1)

Name of test substance: SH-0850

Test period

From November 27, 2012 to November 30, 2012

Presence or absence of metabolic activation

Dose level of test substance

(µg/plate)

Number of reverse mutations (number of colonies)

Base pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Negative control

117

119

123

(120+/-3)

17

11

11

(13+/-3)

31

33

33

(32+/-1)

24

19

19

(21+/-3)

8

14

14

(12+/-3)

313

136

130

146

(137+/-8)

11

11

6

(9+/-3)

32

31

28

(30+/-2)

26

25

19

(23+/-4)

6

19

13

(13+/-7)

625

130

100

128

(119+/-17)

6

12

10

(9+/-3)

34

27

29

(30+/-4)

15

15

20

(17+/-3)

12

13

13

(13+/-1)

1250

163

136

146

(148+/-26)

11

13

12

(12+/-1)

35

22

19

(25+/-9)

19

29

24

(24+/-5)

11

18

19

(16+/-4)

2500

178

132

134

(148+/-26)

11

13

12

(12+/-1)

35

22

19

(25+/-9)

19

29

24

(24+/-5)

11

18

19

(16+/-4)

5000

177

166

146

(163+/-16)

12

6

7

(8+/-3)

28

39

32

(33+/-6)

28

21

19

(23+/-5)

14

14

10

(13+/-2)

+S9 mix

Negative control

122

105

119

(115+/-9)

12

10

6

(9+/-3)

39

33

27

(33+/-6)

27

27

30

(25+/-4)

20

17

11

(16+/-5)

313

109

113

113

(112+/-2)

14

6

12

(11+/-4)

29

18

31

(26+/-7)

39

31

35

(35+/-4)

20

13

15

(16+/-4)

625

132

140

115

(129+/-13)

12

14

12

(13+/-1)

34

20

24

(26+/-7)

33

24

29

(29+/-5)

18

22

15

(18+/-4)

1250

118

108

132

(119+/-13)

15

14

11

(13+/-2)

29

19

33

(27+/-7)

27

22

32

(27+/-5)

14

11

15

(13+/-2)

2500

111

130

129

(123+/-11)

10

8

7

(8+/-2)

34

34

22

(30+/-7)

39

25

28

(31+/-7)

15

13

21

(16+/-4)

5000

115

147

144

(135+/-18)

14

13

8

(12+/-3)

32

29

45

(35+/-9)

28

34

20

(27+/-7)

21

13

12

(15+/-5)

Positive control

Positive control groups for which S9 mix is not necessary

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose (µg/plate)

0.01

0.5

0.01

0.1

0.5

(Number of colonies/plate)

747

717

696

(720+/-26)

311

289

307

(302+/-12)

300

362

299

(320+/-36)

473

513

513

(500+/-23)

1810

1698

1769

(1759+/-57)

Positive control groups for which S9 mix is necessary

Name

2AA

2AA

2AA

2AA

2AA

Dose (µg/plate)

1

2

10

0.5

2

(Number of colonies/plate)

693

701

732

(709+/-21)

170

144

193

(169+/-25)

772

682

689

(714+/-50)

201

217

252

(223+/-26)

119

103

107

(110+/-8)

[Notes]

( ): Mean colony number +/- standard deviation (n=3)

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

NaN3: Sodium azide

2AA: 2-Aminoanthracene

 

Table of the test result (Main test-2)

Name of test substance: SH-0850

Test period

From November 30, 2012 to December 3, 2012

Presence or absence of metabolic activation

Dose level of test substance

(µg/plate)

Number of reverse mutations (number of colonies)

Base pair substitution type

Frame shift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9 mix

Negative control

144

141

141

(142+/-2)

11

12

18

(14+/-4)

26

33

46

(35+/-10)

27

15

21

(21+/-6)

20

19

15

(18+/-3)

313

144

146

164

(151+/-11)

17

12

11

(13+/-3)

46

32

41

(40+/-7)

20

26

28

(25+/-4)

8

17

14

(13+/-5)

625

139

158

151

(149+/-10)

18

14

18

(17+/-2)

32

35

32

(33+/-2)

26

19

32

(26+/-7)

20

13

20

(18+/-4)

1250

152

128

150

(143+/-13)

11

10

14

(12+/-2)

35

35

33

(34+/-1)

21

20

18

(20+/-2)

17

19

19

(18+/-1)

2500

153

179

162

(165+/-13)

21

11

15

(16+/-5)

 

40

34

26

(33+/-7)

29

32

22

(28+/-5)

10

21

14

(15+/-6)

5000

210

217

178

(202+/-21)

13

18

19

(17+/-3)

34

32

38

(35+/-3)

33

24

27

(28+/-5)

20

11

14

(15+/-5)

+S9 mix

Negative control

168

153

129

(150+/-20)

14

17

18

(16+/-2)

27

33

25

(28+/-4)

36

40

26

(34+/-7)

25

29

17

(24+/-6)

313

123

135

144

(134+/-11)

13

7

7

(9+/-3)

45

41

48

(45+/-4)

34

33

33

(33+/-1)

21

29

29

(26+/-5)

625

142

145

135

(140+/-6)

13

6

18

(12+/-6)

33

41

35

(36+/-4)

33

34

31

(33+/-2)

15

21

26

(21+/-6)

1250

132

138

128

(132+/-6)

13

8

14

(12+/-3)

35

29

20

(28+/-8)

40

41

21

(34+/-11)

19

20

17

(19+/-2)

2500

139

133

156

(143+/-12)

7

12

14

(11+/-4)

42

35

31

(36+/-6)

21

35

28

(28+/-7)

12

14

19

(15+/-4)

5000

142

148

145

(145+/-3)

10

11

14

(12+/-2)

28

34

52

(38+/-12)

25

38

34

(32+/-7)

17

24

11

(17+/-7)

Positive control

Positive control groups for which S9 mix is not necessary

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

Dose (µg/plate)

0.01

0.5

0.01

0.1

0.5

(Number of colonies/plate)

786

797

727

(770+/-38)

398

430

403

(410+/-17)

343

401

397

(380+/-32)

431

442

435

(436+/-6)

2084

2138

2149

(2124+/-35)

Positive control groups for which S9 mix is necessary

Name

2AA

2AA

2AA

2AA

2AA

Dose (µg/plate)

1

2

10

0.5

2

(Number of colonies/plate)

899

873

771

(848+/-68)

215

218

214

(216+/-2)

772

739

781

(764+/-22)

255

250

235

(247+/-10)

100

110

99

(103+/-6)

[Notes]

( ): Mean colony number +/- standard deviation (n=3)

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

NaN3: Sodium azide

2AA: 2-Aminoanthracene

 

Historical data

Negative control (mean +/- 3S.D.)

 

-S9 mix

+S9 mix

TA100

TA1535

WP2uvrA

TA98

TA1537

TA100

TA1535

WP2uvrA

TA98

TA1537

Mean

 

Standard deviation

107

 

15

10

 

4

25

 

7

20

 

6

10

 

4

112

 

16

10

 

4

28

 

6

28

 

7

14

 

4

Highest count

 

Lowest count

152

 

62

22

 

1

46

 

4

38

 

2

22

 

1

160

 

64

22

 

1

46

 

10

49

 

7

26

 

2

 

Positive control (mean +/- 3S.D.)

 

-S9 mix

+S9 mix

TA100

TA1535

WP2uvrA

TA98

TA1537

TA100

TA1535

WP2uvrA

TA98

TA1537

Name

AF-2

NaN3

AF-2

AF-2

ICR-191

2AA

2AA

2AA

2AA

2AA

Dose level (µg/plate)

0.01

0.5

0.01

0.1

0.5

1

2

10

0.5

2

Mean

 

Standard deviation

737

 

85

255

 

65

315

 

44

522

 

80

1462

 

243

835

 

115

213

 

34

712

 

125

271

 

40

161

 

24

Highest count

 

Lowest count

992

 

482

450

 

60

447

 

183

762

 

282

2191

 

733

1180

 

490

315

 

111

1087

 

337

391

 

151

233

 

89

[Notes]

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

NaN3: Sodium azide

2AA: 2-Aminoanthracene

Test period: From May, 2012 to October, 2012

Test media: AN medium was used.

Applicant's summary and conclusion

Conclusions:
It was concluded that SH-0850 is not mutagenic under the condition of the study.
Executive summary:

The mutagenic potential of SH-0850 was assessed by the pre-incubation method in the absence and presence of the metabolic activation system (S9 mix) using the Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and the Escherichia coli strain WP2uvrA.

The test result showed that the number of revertant colonies for all tester strains was less than two-fold of the negative control and the mutagenicity was considered to be negative.

Therefore, it was concluded that SH-0850 does not have mutagenic potential.