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Genetic toxicity in vitro

Description of key information

Three mutagenicity studies conducted according to OECD guidelines 471, 473 and 476 were all found to give negative results.

Link to relevant study records

Referenceopen allclose all

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:
2011 -01-26 till 2011-01-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conforming study under GLP without deviations
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
from May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA pKM101, WP2 pKM101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I; experiment II
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar; plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA pKM101, WP2 pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed in the overlay agar in the test tubes from 1000 - 5000 µg/plate. Precipitation was also observed on the incubated agar plates without S9 mix from 333 - 5000 µg/plate in experiment I and at 2500 and 5000 µg/plate in experiment II, and with S9 mix from 1000 - 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA 97, and WP2 pKM101.

Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
 Summary of Results Pre-Experiment/Experiment

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 pKM101

WP2 uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

15 ± 4

9 ± 3

25 ± 2

138 ± 4

170 ± 5

340 ± 10

Untreated

 

 

19 ± 3

10 ± 4

30 ± 5

156 ± 2

224 ± 10

383 ± 34

Test substance

3 µg

 

15 ± 4

8 ± 1

23 ± 5

142 ± 20

187 ± 43

417 ± 18

 

10 µg

 

15 ± 5

8 ± 1

25 ± 4

117 ± 13

180 ± 25

334 ± 25

 

33 µg

 

13 ± 4

8 ± 1

26 ± 3

132 ± 21

147 ± 13

322 ± 18

 

100 µg

 

13 ± 2

7 ± 2

20 ± 2

134 ± 18

151 ± 22

301 ± 20

 

333 µg

 

19 ± 2P

9 ± 1P

28 ± 6P

143 ± 12P

162 ± 21P

397 ± 25P

 

1000 µg

 

13 ± 3P

7 ± 1P

25 ± 8P

158 ± 3P

101 ± 1P

347 ± 43P

 

2500 µg

 

9 ± 1P M

6 ± 1P

21 ± 5P M

106 ± 1P

100 ± 2P

259 ± 7P

 

5000 µg

 

8 ± 2P M

5 ± 1P M

22 ± 4P M

86 ± 10P M

42 ± 9P M

258 ± 25P M

NaN3

10 µg

 

2129 ± 83

 

 

2025 ± 101

 

 

4-NOPD

10 µg

 

 

 

277 ± 20

 

 

 

4-NOPD

50 µg

 

 

76 ± 3

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

2531 ± 197

3035 ± 329

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

18 ± 2

13 ± 3

32 ± 5

126 ± 9

174 ± 6

404 ± 24

Untreated

 

 

24 ± 5

22 ± 2

33 ± 10

145 ± 6

241 ± 29

497 ± 4

Test substance

3 µg

 

20 ± 4

14 ± 7

32 ± 5

124 ± 19

199 ± 23

395 ± 51

 

10 µg

 

20 ± 2

11 ± 4

34 ± 7

134 ± 14

180 ± 35

353 ± 42

 

33 µg

 

14 ± 5

13 ± 2

33 ± 9

113 ± 7

188 ± 20

344 ± 36

 

100 µg

 

16 ± 1

13 ± 3

29 ± 5

132 ± 26

137 ± 4

316 ± 35

 

333 µg

 

16 ± 7

18 ± 2

29 ± 4

147 ± 8

151 ± 3

344 ± 19

 

1000 µg

 

8 ± 2P M

8 ± 2P M

30 ± 6P

121 ± 11P

113 ± 12P

294 ± 32P

 

2500 µg

 

8 ± 2P M

8 ± 2P M

13 ± 2P M

117 ± 3P

105 ± 12P

233 ± 9P

 

5000 µg

 

8 ± 1P M

7 ± 3P M

12 ± 2P M

96 ± 8P M

41 ± 8P M

168 ± 34P

2-AA

2.5 µg

 

392 ± 36

269 ± 40

1731 ± 173

1984 ± 352

 

 

2-AA

10.0 µg

 

 

 

 

 

1412 ± 60

1443 ± 27

 

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

 Summary of Results Experiment II

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 pKM101

WP2 uvrA pKM101

 

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

16 ± 1

10 ± 3

22 ± 6

124 ± 10

183 ± 21

365 ± 20

Untreated

 

 

20 ± 1

13 ± 2

30 ± 1

160 ± 5

202 ± 40

372 ± 18

Test substance

3 µg

 

16 ± 4

11 ± 4

21 ± 1

130 ± 5

199 ± 5

331 ± 30

 

10 µg

 

14 ± 2

11 ± 4

24 ± 3

115 ± 15

185 ± 13

312 ± 10

 

33 µg

 

17 ± 3

10 ± 2

24 ± 2

104 ± 3

178 ± 13

324 ± 25

 

100 µg

 

17 ± 2

13 ± 1

23 ± 4

111 ± 11

177 ± 7

336 ± 19

 

333 µg

 

15 ± 2

14 ± 1

21 ± 1

89 ± 12

173 ± 19

334 ± 21

 

1000 µg

 

10 ± 2

12 ± 4

23 ± 2

78 ± 22

110 ± 4

288 ± 50

 

2500 µg

 

8 ± 1P

7 ± 2P

21 ± 2P

54 ± 8P

93 ± 4P

195 ± 11P

 

5000 µg

 

2 ± 0P M

1 ± 1P M

5 ± 2P M

21 ± 5P M

50 ± 6P M

149 ± 17P M

NaN3

10 µg

 

1735 ± 114

 

 

2196 ± 108

 

 

4-NOPD

10 µg

 

 

 

374 ± 14

 

 

 

4-NOPD

50 µg

 

 

74 ± 9

 

 

 

 

MMS

3.0 µL

 

 

 

 

 

3224 ± 165

2523 ± 147

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

15 ± 7

21 ± 1

34 ± 6

133 ± 22

200 ± 6

397 ± 11

Untreated

 

 

20 ± 6

26 ± 5

44 ± 7

154 ± 12

249 ± 39

412 ± 47

Test substance

3 µg

 

17 ± 7

22 ± 2

37 ± 6

136 ± 10

196 ± 27

373 ± 18

 

10 µg

 

16 ± 6

19 ± 3

29 ± 7

130 ± 7

186 ± 38

379 ± 9

 

33 µg

 

11 ± 3

19 ± 7

31 ± 5

129 ± 18

219 ± 22

368 ± 18

 

100 µg

 

14 ± 1

19 ± 4

36 ± 1

116 ± 12

221 ± 17

369 ± 15

 

333 µg

 

18 ± 2

19 ± 3

33 ± 9

125 ± 15

198 ± 26

358 ± 19

 

1000 µg

 

15 ± 2P

15 ± 5P

35 ± 2P

104 ± 13P

122 ± 4P

332 ± 26P

 

2500 µg

 

12 ± 2P M

11 ± 4P M

22 ± 2P M

84 ± 12P M

77 ± 6P M

233 ± 14P M

 

5000 µg

 

9 ± 3P M

6 ± 2P M

14 ± 1P M

85 ± 6P M

65 ± 11P M

201 ± 22P M

2-AA

2.5 µg

 

296 ± 16

199 ± 1

1447 ± 136

2103 ± 137

 

 

2-AA

10.0 µg

 

 

 

 

 

1459 ± 66

1664 ± 73

 

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

P

M

Precipitate

Manual count

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (μg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

 without S9 mix

 with S9 mix

TA1535

/

2500

5000

/

TA1537

/

/

5000

5000

TA98

/

2500, 5000

5000

5000

TA100

/

/

2500, 5000

/

WP2 pKM101

5000

5000

5000

2500, 5000

WP2 uvrA pKM101

/

5000

5000

/

/ = no toxic effects observed

 
Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5),

were observed at higher concentrations in strain WP2 pKM101 without S9 mix and in strains TA1535, TA98, WP2 pKM101, and WP2 uvrA pKM101 with S9 mix in experiment I. In experiment II, toxic effects were observed without S9 mix in all strains and with S9 mix in strains TA 1537, TA97, and WP2 pKM101.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
According to OECD guideline 473. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
With metabolic activation:
Main Experiment : 32.6, 57.1, 99.9, 174.9, 306.0 µg/mL

Without metabolic activation:
Main Experiment : 25.0, 50.0, 75.0, 400.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
One experiment was performed. The exposure period was 4 hours with and without metabolic activation. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: At least 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The test item, suspended in DMSO, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

One experiment was performed. The exposure period was 4 hours with and without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.

In each treatment group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells per culture were counted for determination of mitotic index.

The highest treatment concentration in the main study, 2870.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (90.3 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.

Precipitation of the test item in the culture medium was observed at 75.0 µg/mL and above at the end of treatment in the absence of S9 mix. In the presence of S9 mix, precipitation of the test item in the culture medium was observed at 57.1 µg/mL and above at the end of treatment. No relevant increase or decrease in the osmolarity or pH value was observed (pre-experiment: solvent control: 353 mOsm, pH 7.6 versus 348 mOsm and pH 7.4 at 2870.0 µg/mL; main experiment: solvent control: 379 mOsm, pH 7.2 versus 375 mOsm and pH 7.2 at 1000.0 µg/mL).

In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (53.3 % of control). In the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.

In the absence of S9 mix, no statistically significant and biologically relevant increase was observed at the concentrations evaluated. The aberration rates of the cells after treatment with the test item (1.0 – 2.5 % aberrant cells, excluding gaps) were close to the solvent control value (1.0 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. In the presence of S9 mix statistically significant increases were observed after treatment with 99.9, 174.9 and 306.0 µg/mL (6.0, 8.5 and 7.5 % aberrant cells, excluding gaps, respectively) clearly exceeding the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Either EMS (825.0 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study

Preparation
interval

Test item
concentration

Mitotic indices

Aberrant cells

 

 

in µg/mL

in %

in %

 

 

 

of control

incl. gaps*

excl. gaps*

carrying exchanges

Exposure period 4 hrs without S9 mix

22 hrs

Solvent control1

100.0

1.0

1.0

0.0

 

 

Positive control2

82.0

14.0

14.0S

3.5

 

 

25.0

86.1

2.0

2.0

0.0

 

 

50.0

90.9

2.5

2.5

0.0

 

 

75.0P

78.9

1.5

1.0

0.0

 

 

400.0P

53.3

3.0

2.5

0.0

Exposure period 4 hrs with S9 mix

22 hrs

Solvent control1

100.0

2.5

2.0

0.0

 

 

Positive control3

57.0

12.0

11.0S

2.0

 

 

32.6

90.2

2.0

2.0

0.0

 

 

57.1P

82.6

2.0

2.0

0.0

 

 

99.9P

103.0

7.0

6.0S

1.0

 

 

174.9P

116.6

8.5

8.5S

0.0

 

 

306.0P#

110.2

7.5

7.5S

0.3

*      Including cells carrying exchanges

#       Evaluation of 200 metaphases per culture

P      Precipitation occurred at the end of treatment

S      Aberration frequency statistically significant higher than corresponding control values

1       DMSO     0.5 % (v/v)

2         EMS    825.0 µg/mL

3       CPA       15.0 µg/mL

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation, when tested up to precipitating concentrations.
Executive summary:

Thisin vitro assay was performed to assess the potential of the test substance to induce structural chromosomal aberrations in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/b-naphthoflavone treated male rats).

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were scored for structural chromosomal aberrations.

 

The highest applied concentration in the main study (2870.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (90.3 %) of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and test item precipitation and in accordance with OECD Guideline 473. 

In the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. 

 

In the absence of S9 mix, no clastogenicity was observed at the concentrations evaluated. In the presence of S9 mix statistically significant increases above the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) were observed with 99.9, 174.9 and 306.0 µg/mL. 

 

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Conducted according to OECD 476. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 4 hours treatment with and without metabolic activation
third experiment: 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 11.3; 22.5; 45.0; 90.0; 135.0; and 180.0 µg/mL
with S9 mix: 2.8; 5.6; 11.3; 22.5; 33.8; and 45.0 µg/mL

Experiment II:
without S9 mix: 22.5; 45.0; 90.0; 135.0; 180.0; and 360.0 µg/mL
with S9 mix: 5.6; 11.3; 22.5; 33.8; 45.0; and 67.5 µg/mL
Experiment III:
with S9 mix: 5.0; 10.0; 30.0; 50.0; 70.0; 90.0; and 110.0 µg/mL
Following the expression phase of 48 hours the cultures at 11.3 µg/mL in experiment I without metabolic activation and at 2.8 mg/mL with metabolic activation were not continued since a minimum of only four analysable concentrations is required by the test guidelines. In experiment II the cultures at 5.6 µg/mL with metabolic activation were not continued for the same reason. The cultures at 360 µg/mL in experiment II without metabolic activation were not continued due to exceedingly severe toxic effects. In experiment III the cultures at the maximum concentration of 110.0 µg/mL were not continued for the same reason. The cultures at the lowest concentration of 5.0 mg/ml were not continued since a minimum of only four analysable concentrations is required by the test guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and low toxicity to the cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above
the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (solvent control: pH 7.47 versus pH 7.51 at 2880 µg/mL)
- Effects of osmolarity: (solvent control: 373 mOsm, versus 339 mOsm at 2880 µg/mL)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation:
Pre-test:
The test medium was checked for precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation
occurred at 90.0 µg/mL and above without metabolic activation and at 180 µg/mL and above with metabolic activation

Main experiments:
The test medium was checked for precipitation visible to the naked eye at the end of the treatment period just before the test item was removed. Precipitation meeting the criteria mentioned above was noted in the pre-experiment at 90.0 µg/mL and above without metabolic activation and at 180.0 µg/mL and above with metabolic activation. In experiments I and II precipitation was determined at 135.0 µg/mL and above in the absence of metabolic activation. In experiment III precipitation was noted at 110.0 µg/mL

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence and absence of metabolic activation with a treatment time of 4 hours. Test item concentrations between 22.5 µg/mL and 2880 µg/mL equal to approximately 10 mM were used. The highest concentration in the pre-experiment was chosen with regard to the molecular weight of the test item (259.1 g/mol) and the purity of 90.3 % w/w.

Cytotoxic effects leading to RSG values below 50% were observed at 180.0 µg/mL and above in the absence of metabolic activation. In the presence of metabolic activation relevant cytotoxic effects occurred down to the lowest concentration of 22.5 µg/mL.

The test medium was checked for precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 90.0 µg/mL and above without metabolic activation and at 180 µg/mL and above with metabolic activation.

Both, pH value and osmolarity was determined in the pre-experiment at the maximum concentration of the test item and at the solvent control without metabolic activation. No relevant increase of the osmolarity or pH value was observed (solvent control: 373 mOsm, pH 7.47 versus 339 mOsm and pH 7.51 at 2880 µg/mL).

The dose range of the main experiments was limited by cytotoxic effects or precipitation. An additional experiment (experiment III) was performed to cover the cytotoxic range of approximately 10-20% relative cloning efficiency I or relative total growth that was not reached by both parallel cultures in experiments I and II.

To overcome problems with possible deviations in toxicity or solubility the main experiments were started with more than four concentrations. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic or precipitating range more closely. Very narrow spacing was used in experiment III to closely cover the cytotoxic range with both parallel cultures.



COMPARISON WITH HISTORICAL CONTROL DATA: Supplies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency 1 (survival) or a relative total growth (RTG) of less than 50% in both parallel cultures occurred in the first experiment without metabolic activation. In the second experiment without metabolic activation cytotoxicity was noted at 180 µg/mL. The next higher concentration of 360 µg/mL was not analysed due to exceedingly severe toxic effects and heavy precipitation. So, the maximum analysable concentration without metabolic activation was limited by the solubility of the test item in aqueous media. In the presence of metabolic activation cytotoxic effects as described above were observed at 33.8 µg/mL and above in experiment I and at 67.5 µg/mL in experiment II. In experiment III relevant toxic effects occurred at 30.0 µg/mL and above. The recommended cytotoxic range of approximately 10-20% RTG was covered in culture I of the first experiment and in culture II of the second experiment with metabolic activation. In the third experiment, performed solely with metabolic activation the recommended toxic range was covered from 50.0 to 90.0 µg/mL in culture I and from 30.0 to 50.0 µg/mL in culture II.
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Y cells
Remarks:
Migrated from field 'Test system'.
Summary Table
      relative relative mutant   relative relative mutant  
  conc. µg S9 cloning total colonies/   cloning total colonies/  
  per mL mix efficiency 1 growth 106cells threshold efficiency 1 growth 106cells threshold
Column 1 2 3 4 5 6 7 8 9 10
Experiment I / 4 h treatment culture I            culture II      
Solv. control with DMSO - 100.0 100.0 112 238 100.0 100.0 104 230
Pos. control with MMS 19.5 - 115.3  34.9 427 238  94.4  38.7 376 230
Test item  11.3 - 112.8 culture was not continued# 100.0 culture was not continued#
Test item  22.5 -  98.1  75.3 112 238 110.4 103.2 146 230
Test item  45.0 -  88.2  76.4 128 238  54.8  92.3  76 230
Test item  90.0 -  93.0  88.3 108 238 106.6  72.2 117 230
Test item 135.0 (p) -  85.3  59.5 131 238 103.1  67.3  98 230
Test item 180.0 (p) -  81.3  35.8 145 238  92.0  53.4 149 230
Solv. control with DMSO + 100.0 100.0 111 237 100.0 100.0  91 217
Pos. control with CPA   3.0 +  97.2  62.6 438 237  60.9  40.3 437 217
Pos. control with CPA   4.5 +  54.8  34.1 741 237  43.6  27.6 686 217
Test item   2.8  +   91.9 culture was not continued#  94.0 culture was not continued#
Test item   5.6  +   83.2  41.8 120 237  76.4  49.3 116 217
Test item  11.3  +   73.1  52.0 111 237  65.4  72.0  66 217
Test item  22.5  +   80.8  54.5 104 237  56.7  52.7  99 217
Test item  33.8  +   55.6  19.0 136 237  57.5  44.0 108 217
Test item  45.0  +   53.9  26.2 182 237  27.3  34.9 112 217
Experiment II / 4 h treatment culture I            culture II      
Solv. control with DMSO - 100.0 100.0  90 216 100.0 100.0  97 223
Pos. control with MMS 19.5 -  97.2  33.0 348 216  58.1  26.9 472 223
Test item 22.5 -  95.8  94.8 101 216  95.1  69.8 162 223
Test item 45.0 -  75.2 107.3  78 216  89.1  44.7 264 223
Test item 90.0 -  79.7  65.1  99 216  87.7  75.6 110 223
Test item 135.0 (p) -  44.6  68.2  97 216  72.0  61.7 102 223
Test item 180.0 (p) -  34.9  58.6 110 216  76.4  46.5 124 223
Test item 360.0 (p) -  8.3 culture was not continued##  27.6 culture was not continued##
Solv. control with DMSO + 100.0 100.0 107 233 100.0 100.0 71 197
Pos. control with CPA 3.0 + 80.6 86.0 160 233 84.8 67.7 221 197
Pos. control with CPA 4.5 + 49.9 45.5 296 233 57.9 45.8 256 197
Test item 5.6 + 52.8 culture was not continued# 59.6 culture was not continued#
Test item 11.3  +  73.6 55.7 110 233 70.8 59.5 78 197
Test item 22.5  +  93.3 79.4 117 233 70.8 50.9 99 197
Test item 33.8  +  78.2 57.9 122 233 66.8 42.8 92 197
Test item 45.0  +  78.2 44.1 113 233 54.0 50.4 137 197
Test item 67.5  +  60.9 46.8 113 233 64.0 24.6 156 197
Experiment III / 4 h treatment culture I            culture II      
Solv. control with DMSO + 100.0 100.0 107 233 100.0 100.0  94 220
Pos. control with CPA 3.0 +  73.1  74.5 142 233  81.6  58.3  95 220
Pos. control with CPA 4.5 +  50.9  42.1 256 233  48.9  18.9 295 220
Test item 5.0 +  72.0 culture was not continued#  75.1 culture was not continued#
Test item 10.0 +  78.9  77.1  96 233 100.0  35.3 110 220
Test item 30.0 +  33.4  35.0 124 233  48.2  16.5  96 220
Test item 50.0 +  24.4  24.3 131 233  35.7  10.7 143 220
Test item 70.0 +  19.1  17.9 111 233  27.1  6.8  96 220
Test item 90.0 +  9.5  9.1 117 233  11.6  5.8 106 220
Test item 110.0 (p) +  4.5 culture was not continued##  5.2 culture was not continued##

threshold = number of mutant colonies per 106cells of each solvent control plus 126

p   precipitation

#   culture was not continued since a minimum of only four analysable concentrations is required by the guidelines

## culture was not continued due to strong toxic effects


Conclusions:
Interpretation of results (migrated information):
negative

The test substance is considered to be non mutagenic in this mouse lymphoma assay.
Executive summary:

The study was performed to investigate the potential of CA4915 to induce mutations at the mouse lymphoma thymidine kinase locus of the cell line L5178Y. 

      

The maximum concentration of 2880 µg/mL equal to approximately 10 mM in the pre-experiment was chosen with regard to the molecular weight of the test item (259.1 g/mol) and the purity (90.3 % w/w). The concentration range of the main experiments was limited by cytotoxicity and precipitation and they were evaluated at the concentrations shown.

 

Experiment I:

without S9 mix:                                   22.5; 45.0; 90.0; 135.0; and 180.0 µg/mL
with S9 mix:                                              5.6; 11.3; 22.5; 33.8; and 45.0 µg/mL

Experiment II:

without S9 mix:                                   22.5; 45.0; 90.0; 135.0; and 180.0 µg/mL
with S9 mix:                                            11.3; 22.5; 33.8; 45.0; and 67.5 µg/mL

Experiment III:

with S9 mix:                                            10.0; 30.0; 50.0; 70.0; and 90.0 µg/mL

Relevant cytotoxic effects occurred in the main experiments with metabolic activation.

 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximum concentration with and without metabolic activation.

 

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

The assay was performed in three independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours. Experiment III was solely performed with microsomal activation and a treatment period of 4 hours. 

     

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reportedCA4915did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test (bacterial reverse mutation in vitro): Negative

A key study conducted according to guideline OECD 471 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Chromosome aberration: Negative

A key study conducted according to guideline OECD 473 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.

Cell mutation assay: Negative

A key study conducted according to guideline OECD 476 and under GLP is available. The study is considered to be relevant, reliable (Klimisch 1) and adequate for the purposes of risk assessment, classification and labeling.


Justification for classification or non-classification

Three mutagenicity studies conducted according to OECD guidelines 471, 473 and 476 were all found to give negative results. As a result, and in accordance with Directive 2001/59/EC, Annex VI, 4.2.2.3, the substance is not considered to be classified as mutagenic.

Three mutagenicity studies conducted according to OECD guidelines 471, 473 and 476 were all found to give negative results. As a result, and in accordance with Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5.2, the substance is not considered to be classified as mutagenic.