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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro reverse gene mutation in bacteria (Ames):

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA100 and TA98) as indicator strains and the liver microsome fraction of rats for metabolic activation system (S9 Mix).

The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The number of revertant colonies of BS-1 did not show any tendency to increase twice or more over that in negative control

for all strains. It was confirmed that the test was appropriately performed because the negative control and the

positive control induced the reasonable increase in the number of revertant colonies.

Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA1535 and TA1537), Escherichia coli (WP2uvrA/pKM101) as indicator strains and the liver microsome

fraction of rats for metabolic activation system (S9 Mix). The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety

and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The number of revertant colonies of BS-1 increased twice or more over that in negative control in TA1535+S9Mix at main test. But this response is not reproducible because the number of revertant colonies did not increase more than double of spontaneous reversion at dose range finding test and confirmatory test-1 and -2. Thus, it is thought that the number of revertant colonies in TA1535+S9Mix at main test increased because of variability. For the other strains, the number of revertant colonies did not show any tendency to increase twice or more over that in negative control. It was confirmed that the test was appropriately performed because the negative control and the positive control induced the reasonable increase in the number of revertant colonies.

Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.

In-vitro cytogenicity in mammalian cells (chromose aberration)

 Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1990). 

 

 Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the in the absence of any marked toxicity the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test item BHES (µg/mL)

4(20)-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

4(20)-hour with S9 (2%)

48.13, 96.25, 192.5, 385, 770, 1540

24-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.

 

Conclusion

The test item,BHES was considered to be non-clastogenic to human lymphocytes in vitro.

In-vitro gene mutation in mammalian cells (Mouse Lymphoma Assay):

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group

Concentration of BHES (µg/mL) plated for mutant frequency

4-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

4-hour with S9 (2%)

24-hour without S9

 Results……..

With no evidence of precipitate or test item-induced toxicity, the maximum dose level used in the Mutagenicity Test was the 10mM limit dose of 1540 µg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 2 September 1997 and 16 September 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law"
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test substance: BS-1
Sample nurnber: 97-180
Concentration: 65 percent aqueous solution
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The highest dose of the test solution was 10μl because the concentration of test substance was 65%.
Solutions of five different concentration, 0. 391, 0. 156, 0. 6 25, 2. 50 and 10. 0 μl per per plate were used for dose range finding test.
For main test, concentration of the test solution was prepared to be 0. 6 25, 1.25, 2.50, 5.00 and 10.0 μl per plate.
Vehicle / solvent:
sterilized pure water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO. 0.01 and 0.1µg/plate for TA100 and TA98
Positive control substance:
other: 2- ( 2-Furyl)-3- (5-nitro- 2-furyl)acrylamide (AF2)
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
dissolved in DMSO. 5 µg/plate for TA100 and TA98
Positive control substance:
other: Benzo[a]pyrene (BP)
Remarks:
with S9 mix
Details on test system and experimental conditions:
Control test
For the solvent control test, three plates were used.
Meanwhile, two plates were used for positive control test.
Every positive control was dissolved in DMSO and stored in a freezer at - 20 °C.

For the test substance two plates of each dose were used in every test.

Test method
Test solution (0.1 ml) was mixed with 0.1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of tester strain (0.1 ml) and then the mixture was pre-incubated at
37 °C for 20 minutes with gentle shaking. For metabolic activation, S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar ( 2 ml)
was added and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0.5 mM D-biotin and 0.5 mM L-histidine.
After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control were obtained.
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
Positive controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
Dose range finding test:
The number of revertant colonies did not increase twice or more over that in negative control in test strains both with and without S9Mix.

Main test:
The number of revertant colonies did not increase twice or more over that in negative control in test strains both with and without S9Mix.

Results from the main test are presented in the table below

Test period

97/9/11~97/9/16

With (+) or without (-) S9 mix

Dose µg/plate

Number of revertant colonies /plate

Base-pair substitution type

Frameshift type

TA100

TA98

 

 

 

 

 

 

S9 Mix (-)

Solvent control

127

 

18

 

167

 

28

 

140

(145)

23

(23)

1      0.625

136

 

25

 

145

(141)

14

(20)

2        1.25

155

 

19

 

127

(141)

23

(21)

3          2.5

140

 

18

 

140

(140)

26

(22)

4              5

144

 

14

 

128

(136)

23

(19)

5           10

137

 

21

 

142

(140)

23

(22)

 

 

 

 

 

 

S9 Mix (+)

Solvent control

139

 

38

 

116

 

48

 

127

(127)

40

(42)

1      0.625

112

 

39

 

161

(137)

43

(41)

2        1.25

122

 

40

 

145

(134)

40

(40)

3          2.5

155

 

37

 

130

(143)

40

(39)

4              5

136

 

28

 

148

(142)

44

(36)

5           10

133

 

27

 

155

(144)

40

(34)

Positive control without S9 mix

Chemical

AF2

AF2

Dose µg/plate

0.01

0.1

Revertants /plate

1166

1240

 

(1203)

164

140

 

(152)

Positive control with S9 mix

Chemical

BP

BP

Dose µg/plate

5

5

Revertants /plate

1122

1036

 

(1079)

293

367

 

(330)

The figures in parentheses show the mean of each plate.

Positive controls

AF2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

BP: Benzo[a]pyrene

Conclusions:
The number of revertant colonies of BS-1 did not show any tendency to increase twice or more over that in negative control
for all strains. It was confirmed that the test was appropriately performed because the negative control and the
positive control induced the reasonable increase in the number of revertant colonies.
Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.
Executive summary:

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA100 and TA98) as indicator strains and the liver microsome fraction of rats for metabolic activation system (S9 Mix).

The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of Manufacture, etc. , of Chemical Substance".

The test substance was judged non-mutagenic in this test system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 17 February 1998 and 12 March 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety and Health Law"
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Test Substance: BS-1
Sample number: 97-180
Concentration: 65 percent aqueous solution
Target gene:
histidine or tryptophan
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The highest dose of the test solution was 10μl because the concentration of test the substance was 65%. Solutions of five
different concentration, 0.391, 0.156 , 0.625, 2.50 and 10.0 μl per plate were used for dose range finding test.
For main test and confirmatory test for TA1535 + S9 Mix, concentration of the test solution was prepared to be 0. 625, 1.25, 2.50, 5.00 and 10.0 μl
per plate. Solutions of five different concentration, 0.156, 0.313, 0.625, 1.25 and 2.50 μl per plate were used for confirmatory test for TA1537 - S9 Mix.
Vehicle / solvent:
sterilized pure water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO. 0.01 µg/plate for strain WP2uvrA/pKM101
Positive control substance:
other: 2-(2-Furyl)-3- (5-nitro-2-furyl)acrylamide (AF2)
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO. 0.5 µg/plate for strain TA1535
Positive control substance:
sodium azide
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterlized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO. 80 µg/plate for strain TA1537
Positive control substance:
9-aminoacridine
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO. Dissolved in DMSO. 2 µg/plate for strain TA1535 and 1 µg/plate for strain WP2urvA/pKM101
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized pure water
True negative controls:
no
Positive controls:
yes
Remarks:
Dissolved in DMSO. 5 µg/plate for strain TA1537
Positive control substance:
benzo(a)pyrene
Remarks:
with S9
Details on test system and experimental conditions:
Control test
To confirm the activity of strains and 89 Mix, solvent control tests and positive control tests were carried out with
and without S9 Mix for each strain.
For the solvent control test, three plates were used. Meanwhile, two plates were used for positive control test.

For the test substance two plates of each dose were used in every test.

Test method
Test solution (0.1ml) was mixed with 0. 1 M sodium phosphate buffer (pH 7.4, 0.5 ml) and the overnight culture of
tester strain (0.1 ml) and then the mixture was pre-incubated at 37 °C for 20 minutes with gentle shaking. For metabolic
activation, S9 Mix (0.5 ml) was mixed instead of 0.1 M sodium phosphate buffer. After pre-incubation, melted soft agar (2 ml)
was added and the resulting mixture was poured onto minimal glucose agar plate. The soft agar contained 0. 5 mM D-biotin and
0. 5 mM L-histidine for Salmonella typhimurium strains, and 0. 5mM L-tryptophan for Escherichia coli strain.
After incubation for 2 days, revertant colonies were counted and average number of colonies in each dose and control
were obtained.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In the initial test (+ S) the number of revertent colonies increased twice or more over that in the solvent control. However, two confirmatory tests were carried out where the number of revertent colonies did not increase twice or more over the control.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose range finding test:
Although a slight increase in the number of revertant colonies was observed in TA1535+S9Mix and TA1537-S9Mix, the
twofold increase beyond spontaneous reversion was not shown in all test strains.

Main test:
The number of revertant colonies increased twice or more over that in negative control in TA1535+S9Mix. In other strains,
the number of revertant colonies did not show any tendency to increase though a slight increase in the number of revertant
colonies was observed in TA1537-S9Mix. Therefore, confirmatory test-1 and -2 were performed for TA1535+S9Mix and TA1537-S9Mix

Confirmatory test-1 and -2:
The number of revertant colonies did not increase twice or more over that in negative control in test strains.

The results of the main test are presented in the table below

Test period

                    98/02/25~98/02/27

With (+) or without (-) S9 mix

Dose µg/plate

Number of revertant colonies /plate

Base-pair substitution type

Frameshift type

TA1535

WP2urvA/pKM101

TA1537

 

 

 

 

 

 

S9 Mix (-)

Solvent control

8

 

66

 

5

 

8

 

80

 

5

 

16

(11)

62

(69)

5

(5)

1      0.625

12

 

77

 

7

 

17

(15)

85

(81)

11

(9)

2        1.25

7

 

70

 

10

 

14

(11)

92

(81)

8

(9)

3          2.5

13

 

55

 

4

 

16

(15)

71

(63)

9

(7)

4              5

18

 

68

 

4

 

16

(17)

70

(69)

10

(7)

5           10

11

 

48

 

7

 

12

(12)

77

(63)

9

(8)

 

 

 

 

 

 

S9 Mix (+)

Solvent control

8

 

87

 

12

 

7

 

92

 

8

 

9

(8)

97

(92)

15

(12)

1      0.625

12

 

72

 

16

 

9

(11)

92

(82)

10

(13)

2        1.25

13

 

62

 

13

 

17

(15)

82

(72)

10

(12)

3          2.5

15

 

71

 

8

 

18

(17)

89

(80)

8

(8)

4              5

10

 

94

 

15

 

19

(15)

80

(87)

9

(12)

5           10

15

 

90

 

8

 

12

(14)

86

(88)

12

(10)

Positive control without S9 mix

Chemical

NaN3

AF2

9AA

Dose µg/plate

0.5

0.01

80

Revertants /plate

241

244

 

(243)

2042

1956

 

(1999)

402

338

 

(370)

Positive control with S9 mix

Chemical

2AA

2AA

BP

Dose µg/plate

2

1

5

Revertants /plate

203

216

 

(210)

132

144

 

(138)

161

157

 

(159)

The figures in parentheses show the mean of each plate

Positive controls

AF2: 2-(2-F uryJ)-3-(5-nitro-2-furyl)acrylamide

NaN3: Sodium azide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo[a]pyrene

Results from Confirmatory test 1 are displayed in the following table

Test period

                    98/02/25~98/02/27

With (+) or without (-) S9 mix

Dose µg/plate

Number of revertant colonies /plate

Base-pair substitution type

Frameshift type

TA1535

TA1537

 

 

 

 

 

 

S9 Mix (-)

Solvent control

 

 

3

 

 

 

4

 

 

 

9

(5)

1      0.625

 

 

4

 

 

 

4

(4)

2        1.25

 

 

5

 

 

 

6

(6)

3          2.5

 

 

5

 

 

 

5

(5)

4              5

 

 

2

 

 

 

7

(5)

5           10

 

 

3

 

 

 

2

(3)

 

 

 

 

 

 

S9 Mix (+)

Solvent control

17

 

 

 

22

 

 

 

18

(19)

 

 

1      0.625

15

 

 

 

28

(22)

 

 

2        1.25

17

 

 

 

23

(20)

 

 

3          2.5

21

 

 

 

20

(21)

 

 

4              5

14

 

 

 

31

(23)

 

 

5           10

33

 

 

 

22

(23)

 

 

Positive control without S9 mix

Chemical

 

9AA

Dose µg/plate

 

80

Revertants /plate

 

 

714

512

 

(613)

Positive control with S9 mix

Chemical

2AA

 

Dose µg/plate

2

 

Revertants /plate

350

374

 

(362)

 

 

Results from the Confirmatory test 2 are displayed in the table below

Test period

                    98/02/25~98/02/27

With (+) or without (-) S9 mix

Dose µg/plate

Number of revertant colonies /plate

Base-pair substitution type

Frameshift type

TA1535

TA1537

 

 

 

 

 

 

S9 Mix (-)

Solvent control

 

 

12

 

 

 

14

 

 

 

13

(13)

1      0.625

 

 

11

 

 

 

14

(13)

2        1.25

 

 

6

 

 

 

10

(8)

3          2.5

 

 

9

 

 

 

15

(12)

4              5

 

 

2

 

 

 

9

(6)

5           10

 

 

10

 

 

 

19

(15)

 

 

 

 

 

 

S9 Mix (+)

Solvent control

13

 

 

 

12

 

 

 

11

(12)

 

 

1      0.625

8

 

 

 

13

(11)

 

 

2        1.25

7

 

 

 

10

(9)

 

 

3          2.5

8

 

 

 

12

(10)

 

 

4              5

11

 

 

 

11

(11)

 

 

5           10

8

 

 

 

13

(11)

 

 

Positive control without S9 mix

Chemical

 

9AA

Dose µg/plate

 

80

Revertants /plate

 

 

473

466

 

(470)

Positive control with S9 mix

Chemical

2AA

 

Dose µg/plate

2

 

Revertants /plate

354

404

 

(379)

 

 

Conclusions:
The number of revertant colonies of BS-1 increased twice or more over that in negative control in TA1535+S9Mix at main test. But this response is not reproducible because the number of revertant colonies did not increase more than double of spontaneous reversion at dose range finding test and confirmatory test-1 and -2. Thus, it is thought that the number of revertant colonies in TA1535+S9Mix at main test increased because of variability. For the other strains, the number of revertant colonies did not show any tendency to increase twice or more over that in negative control. It was confirmed that the test was appropriately performed because the negative control and the positive control induced the reasonable increase in the number of
revertant colonies.
Based on the above results, it is concluded that BS-1 was non-mutagenic in this test system.
Executive summary:

BS-1 was tested for mutagenicity using Salmonella typhimurium (TA1535 and TA1537), Escherichia coli (WP2uvrA/pKM101) as indicator strains and the liver microsome

fraction of rats for metabolic activation system (S9 Mix). The test was carried out according to "Standards for Mutagenicity Test using Microorganisms" of "Occupational Safety

and Health Law" and "GLP Standards Applied to Industrial Chemicals" of "Law Concerning the Examination and Regulation of

Manufacture, etc. , of Chemical Substance". The test substance was judged non-mutagenic in this test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 July 2016 and 08 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro Mammalian Chromosome Aberration Test
Specific details on test material used for the study:
Identification: BHES
Physical state/Appearance: White solid block
Batch: 160226
Purity: 99.64%
Expiry Date: 01 March 2017
Storage Conditions: Room temperature in the dark
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability
- Suitability of cells: The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Cell cycle length, doubling time or proliferation index: Average generation time for human lymphocytes is approx. 16 hrs
- Sex, age and number of blood donors if applicable:
Preliminary toxicity test: male, aged 25 years
Main experiment: male, aged 26 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Modal number of chromosomes: 44-48
Metabolic activation:
with and without
Metabolic activation system:
induced rat liver homogenate metabolizing system (S9), at a 2% final concentration
Test concentrations with justification for top dose:
Preliminary test:
The dose range for the Preliminary Toxicity Test was 6.02, 12.03, 24.06, 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL.
The maximum dose was the maximum recommended dose level, the 10 mM concentration.

Main experiment:
In the absence of any marked toxicity in the preliminary test, the maximum dose level selected for the Main Experiment was the maximum recommended dose level, and was 1540µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.
The test concentration were as follows:
4(20)-hour without S9 - 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL
4(20)-hour with S9 - 48.13, 96.25, 192.5, 385, 770 and 1540µg/mL
24-hour without S9 - 48.13, 96.25, 192.5, 385, 770 and 1540 µg/mL
Vehicle / solvent:
The test item was soluble in culture medium (Minimal Essential Medium (MEM)) at 15.4 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/mL for 4-hour exposure 0.05 µg/mL for the 24-hour exposure
Positive control substance:
mitomycin C
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Cells
Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/BNF S9 10/04/16 was used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 48hrs
- Exposure duration: 4 and 24 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24hrs

SPINDLE INHIBITOR: demecolcine (Colcemid 0.1 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analyzed.

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity was not dose related
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mitotic index data for the Main Experiment confirm the qualitative observations of the preliminary test in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups. In the 4(20)-hour exposure group in the absence of S9, no toxicity was observed at the maximum recommended dose level, 1540 µg/mL. In the presence of S9, there was very modest toxicity demonstrated at 770 µg/mL and 1540 µg/mL with 17% and 10% mitotic inhibition, respectively. The 24-hour exposure group did demonstrate marked toxicity with 53% and 50% mitotic inhibition at 192.5 µg/mL and 770 µg/mL, respectively. However, it was considered that these reductions were due to a discrepancy in the mitotic index between the ‘A’ and ‘B’ replicates, with the ‘B’ replicates being much lower than the ‘A’ replicates. The toxicity demonstrated in the 24-hour exposure group was not dose related and the reductions in mitotic index at 192.5 µg/mL and 770 µg/mL were considered not to be a true representation of the toxicity in this exposure group. An inhibition of mitotic index of 39% was noted at 1540 µg/mL in the 24-hour continuous exposure group.




Preliminary Toxicity Test:

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1540 µg/mL in all three exposure groups. The test item induced no marked evidence of toxicity in any of the exposure groups.  

In the absence of any marked toxicity the maximum dose level selected for the Main Experiment was the maximum recommended dose level, and was 1540µg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.

Chromosome Aberration Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of BHES (µg/mL)

4(20)-hour without S9

0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, MMC0.2*

4(20)-hour with S9 (2%)

0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, CP2*

24-hour without S9

0*, 48.13, 96.25, 192.5*, 385*, 770*, 1540*, MMC 0.05*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 1540 µg/mL in all three exposure groups.

No precipitate of the test item was observed in the blood cultures at the end of the exposure in any of the three exposure groups.

The maximum dose level selected for metaphase analysis was the maximum recommended dose level, the 10 mM concentration dose level (1540 µg/mL).

The assay was considered valid as it met all of the following criteria:

- The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

- All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

- The required number of cells and concentrations were analyzed.

- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure Without Metabolic Activation (S9)

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

            Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

 X

 (+ Gaps)

 (-Gaps)

 (+Gaps)

 (-Gaps)

Vehicle Control (MEM)

A

5.45

150

0

0

0

0

0

0

0

0

0

0

B

5.35

150

0

0

0

0

0

0

0

0

0

0

Total

10.80

300

0

0

0

0

0

0

0

0

0

0

 

(100)

 

 

 

 

 

 

 

 

 

(0.0)

(0.0)

 

A

4.65

150

1

0

0

0

0

0

1

0

1

0

192.5

B

5.85

150

2

0

0

1

0

0

3

1

3

1

mg/mL

Total

10.50

300

3

0

0

1

0

0

4

1

4

1

 

 

(97)

 

 

 

 

 

 

 

 

 

(1.3)

(0.3)

 

A

6.50

150

0

0

0

0

0

0

0

0

0

0

385

B

4.85

150

0

1

0

1

0

0

2

2

2

2

mg/mL

Total

11.35

300

0

1

0

1

0

0

2

2

2

2

 

 

(105)

 

 

 

 

 

 

 

 

 

(0.7)

(0.7)

 

A

5.40

150

1

0

0

2

0

0

3

2

2

1

770

B

6.80

150

2

0

0

0

0

0

2

0

2

0

mg/mL

Total

12.20

300

3

0

0

2

0

0

5

2

4

1

 

 

(113)

 

 

 

 

 

 

 

 

 

(1.3)

(0.3)

 

A

4.60

150

0

0

0

0

0

0

0

0

0

0

1540

B

6.85

150

0

0

0

0

0

0

0

0

0

0

mg/mL

Total

11.45

300

0

0

0

0

0

0

0

0

0

0

 

 

(106)

 

 

 

 

 

 

 

 

 

(0.0)

(0.0)

Positive Control

A

3.70

86a

0

5

8

2

0

0

15

15

15

15

MMC 0.2

B

4.25

133a

2

16

4

0

0

0

22

20

17

17

mg/mL

Total

7.95

199

2

21

12

2

0

0

37

35

32

32***

 

 

(74)

 

 

 

 

 

 

 

 

 

(16.1)

(15.6)

MMC     Mitomycin C

a             Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***          P < 0.001

MEM Minimal Essential Medium

Results of Chromosome Aberration Test – Main Experiment 4(20)-hour Exposure With Metabolic Activation (2% S9)

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

            Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

 X

 (+ Gaps)

 (-Gaps)

 (+Gaps)

 (-Gaps)

Vehicle Control (MEM)

A

5.25

150

3

0

0

0

0

0

3

0

3

0

B

5.50

150

0

2

0

0

0

0

2

2

1

1

Total

10.80

300

3

2

0

0

0

0

5

2

4

1

 

(100)

 

 

 

 

 

 

 

 

 

(1.3)

(0.3)

 

A

5.00

150

1

2

0

0

0

0

3

2

3

2

192.5

B

5.30

150

0

0

0

0

0

0

0

0

0

0

mg/mL

Total

10.50

300

1

2

0

0

0

0

3

2

3

2

 

 

(96)

 

 

 

 

 

 

 

 

 

(1.0)

(0.7)

 

A

5.95

150

1

0

0

0

0

0

1

0

1

0

385

B

5.75

150

0

0

0

0

0

0

0

0

0

0

mg/mL

Total

11.35

300

1

0

0

0

0

0

1

0

0

0

 

 

(109)

 

 

 

 

 

 

 

 

 

(0.3)

(0.0)

 

A

4.10

150

0

1

0

0

0

0

1

1

1

1

770

B

4.85

150

0

0

0

0

0

0

0

0

0

0

mg/mL

Total

12.20

300

0

1

0

0

0

0

1

1

1

1

 

 

(83)

 

 

 

 

 

 

 

 

 

(0.3)

(0.3)

 

A

4.45

150

0

0

0

0

0

0

0

0

0

0

1540

B

5.20

150

1

0

0

0

0

0

1

0

1

0

mg/mL

Total

11.45

300

1

0

0

0

0

0

1

0

1

0

 

 

(90)

 

 

 

 

 

 

 

 

 

(0.3)

(0.0)

Positive Control

A

2.40

40a

1

18

2

1

0

0

22

21

16

15

MMC 0.2

B

2.65

61a

4

19

1

2

0

0

26

22

19

16

mg/mL

Total

7.95

101

5

37

3

3

0

0

48

43

35

31***

 

 

(47)

 

 

 

 

 

 

 

 

 

(34.7)

(30.7)

CP         Cyclophosphamide

a            Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***        P < 0.001

MEM Minimal Essential Medium

Results of Chromosome Aberration Test – Main Experiment 24-hour Continuous Exposure
Without Metabolic Activation (S9)

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

 X

 (+ Gaps)

 (-Gaps)

 (+Gaps)

 (-Gaps)

Vehicle Control (MEM)

A

3.15

150

1

0

0

0

0

0

1

0

1

0

B

3.90

150

0

1

0

0

0

0

1

1

1

1

Total

7.05

300

1

1

0

0

0

0

2

1

2

1

 

(100)

 

 

 

 

 

 

 

 

 

(0.7)

(0.3)

 

A

2.05

150

2

0

0

2

0

0

4

2

3

2

192.5

B

1.25

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

3.30

300

2

0

0

2

0

0

4

2

3

2

 

 

(47)

 

 

 

 

 

 

 

 

 

(1.0)

(0.7)

 

A

3.05

150

1

4

0

0

0

0

5

4

5

4

385

B

2.85

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

5.90

300

1

4

0

0

0

0

5

4

5

4

 

 

(84)

 

 

 

 

 

 

 

 

 

(1.7)

(1.3)

 

A

2.40

150

0

2

0

0

0

0

2

2

2

2

770

B

1.11

150

1

2

0

0

0

0

3

2

3

2

µg/mL

Total

3.51

300

1

4

0

0

0

0

5

4

5

4

 

 

(50)

 

 

 

 

 

 

 

 

 

(1.7)

(1.3)

 

A

1.95

150

0

0

0

0

0

0

0

0

0

0

1540

B

2.35

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

4.30

300

0

0

0

0

0

0

0

0

0

0

 

 

(61)

 

 

 

 

 

 

 

 

 

(0.0)

(0.0)

Positive Control

A

1.20

105a

5

13

0

4

0

0

22

17

19

15

MMC 0.05

B

1.10

95a

2

19

4

3

0

0

28

26

16

15

µg/mL

Total

2.30

200

7

32

4

7

0

0

50

43

35

30***

 

 

(33)

 

 

 

 

 

 

 

 

 

(17.5)

(15.0)

MMC     Mitomycin C

a            Slide evaluation terminated when 15 cells with aberrations (excluding gaps) had been observed

***        P < 0.001

MEM Minimal Essential Medium


Conclusions:
BHES did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

 Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1990). 

 Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the in the absence of any marked toxicity the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test item BHES (µg/mL)

4(20)-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

4(20)-hour with S9 (2%)

48.13, 96.25, 192.5, 385, 770, 1540

24-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.

Conclusion

The test item,BHES was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 01 July 2016 and 26 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian gene mutaation study
Specific details on test material used for the study:
Identification: BHES
Physical state/Appearance: White solid block
Batch: 160226
Purity: 99.64%
Expiry Date: 01 March 2017
Storage Conditions: Room temperature, in the dark
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: The thymidine kinase heterozygote system, TK +/- to TK -/- has been extensively validated
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: The stocks of cells are stored in liquid nitrogen at approximately -196 °C.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes


Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The molecular weight of the test item was 154.18 therefore the maximum proposed dose level in the solubility test was set at 1540 µg/mL, the 10 mM limit dose. The purity of the test item was 99.64% and was therefore not accounted for during formulation of the test item.

Preliminary Cytoxicity test:
0, 6.02, 12.03, 24.06, 48.13, 96.25, 192.5, 385, 770, 1540 µg/mL

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Main experiment:
4 hr exposure (with and without S9) and 24 hr exposure - 0, 48.13, 96.25, 192.5, 385, 770, 1540 µg/mL

Vehicle / solvent:
The test item was accurately weighed and formulated in R0 medium prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
400µg/mL and 150 µg/mL respectively, was used in the 4-hour and 24-hour exposure groups
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 µg/mL was used in the 4-hour exposure group
Positive control substance:
cyclophosphamide
Remarks:
With S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, in suspension
- Cell density at seeding (if applicable):
preliminary toxicty tests 5 x 10^5 cells/ml for 4-hour exposure and 1.5 x 10^5 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 calls/ml after the exposure period, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
Main test: 1 x 10^-6 cells/ml for 4-hour exposure and 0.3 x 10^6 cells/ml for 24-hour exposure. All groups were serially diluted to 2 x 10^5 cells.ml after the exposure period. Before plating cells were diluted to 1 x 10^4 cells/ml (or 10 cells/ml for viability assessment)

DURATION
- Preincubation period: Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment.
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-12 days

SELECTION AGENT (mutation assays): 4 µg/mL 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
Data evaluation:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
To define positive and negative responses the Global Evaluation Factor (GEF) of 126 x 10^-6 is used. This is the increase in mutation frequency (MF) above the concurrent control.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
Calculation of Percentage Relative Suspension Growth (%RSG):
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V):
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = number of negative wells / total wells plated
%V = (-ln P(0) x 100) / number of cells per well

Calculation of Relative Total Growth (RTG):
For each culture, the relative cloning efficiency, RCE, was calculated:
RCE = %V / mean solvent control %V
Finally, for each culture RTG is calculated:
RTG = (RCE x RSG)/100%

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test:
There was no evidence of any marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in any of the three exposure groups. No precipitate of the test item was observed at any of the dose levels.

Mutagenicity Test:
As was seen in the Preliminary Cytotoxicity Test, there was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values. There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had also not occurred. Acceptable levels of toxicity were seen with the positive control substances.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, including the 10 mM limit dose, in any of the three exposure groups. No precipitate of the test item was observed at any of the dose levels.




Preliminary Cytotoxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

6.02

96

93

97

12.03

111

83

113

24.06

94

84

110

48.13

96

92

98

96.25

99

80

83

192.5

96

100

106

385

94

85

89

770

97

82

81

1540

107

96

90

A summary of the results of the main experiment is presented in the tables below:

Concentration

(µg/mL)

4-Hours-S9

Concentration

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

128.30

 

0

 

100

1.00

145.30

 

48.13

 

92

0.94

123.27

 

48.13

 

88

0.93

157.37

 

96.25

 

94

1.04

118.10

 

96.25

 

97

1.05

137.25

 

192.5

 

101

1.22

88.25

 

192.5

 

92

0.98

137.43

 

385

 

101

1.12

113.45

 

385

 

91

1.03

138.74

 

770

 

96

1.14

114.29

 

770

 

96

0.96

157.34

 

1540

 

94

1.12

110.85

 

1540

 

88

0.99

128.05

 

MF threshold for a positive response = 254.30

MF threshold for a positive response = 271.30

Positive control

 

 

Positive control

 

 

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

74

0.62

1030.03

 

1.5

 

73

0.48

1065.80

 

 

 

 

 

 

 

 

 

 

 

 

 

Concentration

(µg/mL)

24-Hours-S9

 

%RSG

RTG

MF§

0

 

100

1.00

158.20

 

48.13

 

109

1.15

123.61

 

96.25

 

124

1.21

157.76

 

192.5

 

96

0.96

160.56

 

385

 

85

0.86

163.92

 

770

 

110

1.03

169.01

 

1540

 

107

1.02

150.35

 

MF threshold for a positive response = 284.20

Positive control

 

 

EMS

 

 

 

 

 

150

 

49

0.39

1482.67

 

A summary of analysis for all test groups is displayed in the following tables:

4-Hour test group (-S9)

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

12.76

100

78.43

1.00

128.30

48.13

 

10.83

92

80.34

0.94

123.27

96.25

 

11.49

94

86.56

1.04

118.10

192.5

 

12.23

101

94.51

1.22

88.25

385

 

12.60

101

87.30

1.12

113.45

770

 

11.77

96

93.66

1.14

114.29

1540

 

11.95

94

93.66

1.12

110.85

Positive control EMS

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

400

 

9.47

74

65.31

0.62

1030.03

 

 

 

 

 

 

 

GEF =126, therefore MF threshold for a positive response = 254.30

4-Hour test group (+S9)

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

10.82

100

73.67

1.00

145.30

48.13

 

9.59

88

78.43

0.93

157.37

96.25

 

11.32

97

80.34

1.05

137.25

192.5

 

9.92

92

79.06

0.98

137.43

385

 

10.06

91

83.01

1.03

138.74

770

 

10.66

96

74.24

0.96

157.34

1540

 

10.52

88

82.33

0.99

128.05

Positive control CP

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

1.5

 

8.24

73

48.70

0.48

1065.80

 

 

 

 

 

 

 

GEF =126, therefore MF threshold for a positive response = 271.30

24-Hour test group (-S9)

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

63.22

100

90.38

1.00

158.20

48.13

 

67.90

109

97.17

1.15

123.61

96.25

 

75.58

124

91.18

1.21

157.76

192.5

 

61.38

96

89.59

0.96

160.56

385

 

55.50

85

88.81

0.86

163.92

770

 

69.24

110

85.11

1.03

169.01

1540

 

65.84

107

88.81

1.02

150.35

Positive control EMS

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

150

 

39.70

49

56.92

0.39

1482.67

 

 

 

 

 

 

 

GEF =126, therefore MF threshold for a positive response = 284.20

A summary of mutation frequencies for all test groups is displayed in the following tables:

4-Hour test group (-S9)

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

160

768

712

768

48.3

684

768

73.8

0.40

48.13

 

77

384

359

384

41.9

340

384

75.7

0.36

96.25

 

68

384

356

384

43.7

341

384

68.6

0.39

192.5

 

58

384

370

384

19.6

339

384

65.9

0.24

385

 

67

384

361

384

35.4

338

384

73.1

0.33

770

 

59

384

355

384

41.9

339

384

66.5

0.39

1540

 

59

384

362

384

31.5

334

384

74.5

0.31

400 EMS

 

104

384

252

384

322.5

232

384

385.8

0.46

4-Hour test group (+S9)

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

176

768

703

768

60.0

685

768

77.6

0.44

48.13

 

80

384

351

384

57.3

333

384

90.8

0.39

96.25

 

77

384

359

384

41.9

333

384

88.7

0.33

192.5

 

79

384

352

384

55.0

341

384

75.1

0.43

385

 

73

384

356

384

45.6

333

384

85.8

0.35

770

 

87

384

348

384

66.3

340

384

82.0

0.45

1540

 

74

384

358

384

42.6

337

384

79.3

0.36

1.5 CP

 

145

384

227

384

539.8

293

384

277.7

0.63

24-Hour test group (-S9)

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

126

768

695

768

55.3

650

768

92.3

0.38

48.13

 

55

384

356

384

39.0

330

384

78.0

0.34

96.25

 

62

384

349

384

52.4

323

384

94.9

0.36

192.5

 

64

384

358

384

39.1

314

384

112.3

0.27

385

 

65

384

353

384

47.4

318

384

106.2

0.32

770

 

70

384

352

384

51.1

320

384

107.1

0.33

1540

 

65

384

347

384

57.0

331

384

83.6

0.41

150 EMS

 

123

384

244

384

398.3

211

384

526.0

0.45

KEY TO TABLES 1 TO 10

$       =       Cell counts (x10^5 cells/mL).  Set up on previous day to 2 x 10^5 cells/mL unless otherwise stated in parenthesis.

%RSG       =       Relative Suspension Growth

RTG       =       Relative Total Growth

%V       =       Viability Day 2

A,B       =       Replicate cultures

CP       =       Cyclophosphamide

EMS       =       Ethylmethanesulphonate

MF§       =       5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Nv       =       Number of wells scored, viability plates

Yv       =       Number of wells without colonies, viability plates

Ym       =       Number of wells without colonies, mutation plates

Nm       =       Number of wells scored, mutation plates

Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group

Concentration of BHES (µg/mL) plated for mutant frequency

4-hour without S9

48.13, 96.25, 192.5, 385, 770, 1540

4-hour with S9 (2%)

24-hour without S9

 Results……..

With no evidence of precipitate or test item-induced toxicity, the maximum dose level used in the Mutagenicity Test was the 10mM limit dose of 1540 µg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on negative results in in-vitro studies conducted on the substance, the substance does not need to be classified for germ cell mutagenicity.