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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 FEB 2010 to 28 JUN 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current testing guidelines and GLP-compliant.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MK92K
- Physical state: brown liquid
- Analytical purity: 75% active ingredient
- Composition of test material, percentage of components: 75% active ingredient, 3.8% propan-2-ol and 21.2% water
- Lot/batch No.: PK231-141/1
- Retest date of the lot/batch: 29 March 2011
- Storage condition of test material: 15 to 25°C in the dark

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium strains: TA98, TA100, TA1535, TA1537 and TA102
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: With the exception of strain TA102, these strains require biotin as well as histidine for growth. In strain TA102 the critical mutation in the histidine gene is located on a multicopy plasmid pAQ1.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Range-finder experiment (with and without): 1.6, 8, 40, 200, 1000 and 5000 µg/plate.
Mutation Experiment 1 (with and without): 0.064, 0.32, 1.6, 8, 40, 200 and 1000 µg/plate.
Mutation Experiment 2 (with and without): 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/plate*.
Mutation experiment 2 (additional treatments of strain TA102, without): 1.95, 3.91, 7.81, 15.6, 31.3, 62.5 and 125 µg/plate.

* (1000 µg/plate concentration tested in strains TA98, TA1535 and TA102 in the absence of S-9 only and all strains in the presence of S-9).
Vehicle / solvent:
Anhydrous analytical grade dimethyl sulphoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene (2NF), Sodium azide (NaN3), 9-aminoacridine (AAC), Mitomycin C (MMC), Benzo[a]pyrene (B[a]P) and 2-aminoanthracene (AAN).
Remarks:
Final plate concentrations (µg/plate): 2NF = 5.0; NaN3 = 2.0; AAC = 50.0; MMC = 0.2; B[a]P = 10.0; AAN = 5.0 or 20.0.
Details on test system and experimental conditions:
Toxicity Range-Finder Experiment
MK92K was tested for toxicity (and mutation) in strain TA100. Triplicate plates without and with S-9 mix were used. Negative (vehicle) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46 ± 1°C:

• 0.1 mL bacterial culture
• 0.1 mL test article solution or control
• 0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 ± 1°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

Mutation Experiments
MK92K was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. Platings were achieved as described above.
Treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S-9 mix detailed above, were mixed together and incubated for 1 hour at 37 ± 1°C, before the addition of 2.5 mL molten agar at 46 ± 1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Following Experiment 2 treatments of TA102 in the absence of S-9, there were insufficient non-toxic concentrations to allow a thorough assessment of mutagenicity. Therefore, additional plate-incorporation treatments were performed at lower test concentrations and these results are presented as Experiment 2 additional data.

Colony counting
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:

1. Dunnett's test gave a significant response (p ≤ 0.01) which was concentration related
2. the positive trend/effects described above were reproducible.

The test article was considered as positive in this assay if all of the above criteria were met.

The test article was considered as negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Individual plate counts from all experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the accepted normal ranges for our laboratory for numbers of spontaneous revertants on vehicle control plates and numbers of induced revertants on positive control plates. Data were considered acceptable if the mean vehicle control counts fell within the historical 99% confidence intervals for group means and/or each vehicle control plate count fell within the historical 99% reference ranges, and the positive control plate counts were comparable with the historical 99% reference ranges. The ranges that are quoted are based on a large volume of historical control data accumulated from experiments where the correct strain and assay functioning are considered to have been confirmed. Data for our laboratory are consistent with ranges of spontaneous revertants per plate considered acceptable elsewhere.
For evaluation of test article and positive control data there are many statistical methods in use, and several are acceptable. Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate).

Results and discussion

Test results
Species / strain:
other: S. typhimurium strains: TA98, TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Evidence of toxicity was observed at 200 and/or 1000 µg/plate and above in the absence or presence of S-9, respectively.
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Summary of mean revertant colonies (-S-9) – Experiment 1

Substance

Dose level µg/plate

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

26 ± 3

113 ± 11

18 ± 5

13 ± 2

330 ± 19

MK92K

0.064

30 ± 6

109 ± 6

15 ± 7

15 ± 2

342 ± 25

0.32

22 ± 4

115 ± 9

21 ± 4

8 ± 4

308 ± 13

1.6

30 ± 3

108 ± 10

16 ± 2

11 ± 5

287 ± 19

8

33 ± 5

122 ± 23

17 ± 3

10 ± 4

348 ± 47

40

25 ± 0

104 ± 13

15 ± 9

12 ± 3

358 ± 34

200

22 ± 6

40 ± 19

15 ± 5

12 ± 6

90 ± 11

1000

T

T

T

T

T

Positive Controls

Compound

2NF

NaN3

NaN3

AAC

MMC

Dose level

5

2

2

50

0.2

Mean ± SD

848 ± 91

720 ± 69

615 ± 105

107 ± 6

896 ± 6

SD         Standard Deviation

T             Toxic, no revertant colonies

 

Table 2: Summary of mean revertant colonies (+S-9) – Experiment 1

Substance

Dose level µg/plate

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

34 ± 5

99 ± 8

16 ± 6

14 ± 5

261 ± 12

MK92K

0.064

36 ± 4

113 ± 20

18 ± 4

17 ± 5

275 ± 23

0.32

34 ± 9

108 ± 10

13 ± 2

22 ± 6

277 ± 13

1.6

37 ± 9

118 ± 13

17 ± 3

25 ± 1

247 ± 20

8

39 ± 2

124 ± 12

19 ± 4

22 ± 3

263 ± 39

40

36 ± 7

103 ± 3

13 ± 3

16 ± 2

276 ± 30

200

44 ± 14

108 ± 10

11 ± 3

19 ± 1

212 ± 32

1000

38 ± 6

25 ± 9.2

9 ± 6

5 ± 5

T

Positive Controls

Compound

B[a]P

AAN

AAN

AAN

AAN

Dose level

10

5

5

5

20

Mean ± SD

364 ± 15

1138 ± 93

217 ± 14

120 ± 51

1875 ± 108

SD         Standard Deviation

T             Toxic, no revertant colonies

 

Table 3: Summary of mean revertant colonies (-S-9) – Experiment 2

Substance

Dose level µg/plate

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

17 ± 5

96 ± 12

13 ± 2

11 ± 4

245 ± 30

MK92K

15.63

22 ± 4

109 ± 2

14 ± 2

10 ± 1

258 ± 32

31.25

20 ± 2

106 ± 7

15 ± 6

11 ± 8

286 ± 10

62.5

25 ± 6

100 ± 6

9 ± 3

10 ± 1

222 ± 17

125

19 ± 7

43 ± 11

16 ± 5

10 ± 3

T

250

8 ± 2

T

8 ± 2

T

T

500

T

T

T

T

T

1000

T

NT

T

NT

T

Positive Controls

Compound

2NF

NaN3

NaN3

AAC

MMC

Dose level

5

2

2

50

0.2

Mean ± SD

675 ± 39

637 ± 22

482 ± 33

90 ± 14

920 ± 98

SD         Standard Deviation

T             Toxic, no revertant colonies

NT          Not Tested

 

Table 4: Summary of mean revertant colonies (+S-9) – Experiment 2

Substance

Dose level µg/plate

TA98

TA100

TA1535

TA1537

TA102

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

Mean ± SD

DMSO

33 ± 8

77 ± 10

15 ± 4

17 ± 7

236 ± 14

MK92K

15.63

35 ± 5

82 ± 16

13 ± 8

19 ± 4

230 ± 14

31.25

35 ± 5

94 ± 12

16 ± 1

22 ± 2

220 ± 5

62.5

31 ± 9

84 ± 4

11 ± 4

15 ± 4

229 ± 9

125

30 ± 8

88 ± 16

18 ± 4

17 ± 2

220 ± 22

250

40 ± 8

87 ± 8

11 ± 5

20 ± 6

205 ± 7

500

38 ± 6

67 ± 9

11 ± 4

14 ± 2

T

1000

17 ± 3

T

T

T

T

Positive Controls

Compound

B[a]P

AAN

AAN

AAN

AAN

Dose level

10

5

5

5

20

Mean ± SD

333 ± 34

626 ± 43

202 ± 6

105 ± 19

1748 ± 57

SD         Standard Deviation

T             Toxic, no revertant colonies

 

Table 5: Summary of mean revertant colonies (-S-9) – Experiment 2 additional treatments

Substance

Dose level µg/plate

TA102

Mean ± SD

DMSO

100 µL

265 ± 14

MK92K

1.95

257 ± 30

3.91

280 ± 5

7.81

297 ± 14

15.63

293 ± 3

31.25

282 ± 20

62.5

205 ± 11

125

T

Positive Controls

Compound

MMC

Dose level

0.2

Mean ± SD

889 ± 10

SD         Standard Deviation

T             Toxic, no revertant colonies

 

 

Applicant's summary and conclusion

Conclusions:
It was concluded that MK92K did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 125 µg/plate (toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

MK92K was assayed for mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254‑induced rat liver post-mitochondrial fraction (S‑9), in two separate experiments.

All MK92K treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO), and all concentrations tested were corrected for 75% active ingredient.

An initial toxicity Range‑Finder Experiment was carried out in the absence and in the presence of S-9 in strain TA100 only, using final concentrations of MK92K at 1.6, 8, 40, 200, 1000 and 5000 mg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 200 or 1000 mg/plate and above in the absence or presence of S-9, respectively. These data were considered to be acceptable for toxicity assessment only as insufficient non toxic concentrations were available to allow a thorough assessment of mutagenic activity.

Experiment 1 treatments of all tester strains were performed in the absence and in the presence of S‑9. The maximum concentration was reduced to 1000mg/plate based on toxicity observed in the Range Finder Experiment and a concentration range of 0.064 ‑ 1000 mg/plate was employed. Following these treatments, evidence of toxicity was observed at 200 and/or 1000 mg/plate in all strains in the absence and presence of S-9.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For strains TA98, TA1535 and TA102 in the absence and presence of S-9, and strains TA100 and TA1537 in the presence of S-9 only, the maximum test concentration was 1000 mg/plate, for strains TA100 and TA1537 in the absence of S-9, the maximum test concentration was reduced to 500 mg/plate based on strain specific toxicity observed in previous experiments. Narrowed concentration intervals were employed covering the ranges 15.63 – 1000 mg/plate (TA98, TA1535 and TA102 in the absence and presence of S-9, and strains TA100 and TA1537 in the presence of S-9 only) or 15.63 – 500 mg/plate (TA100 and TA1537 in the absence of S-9), in order to examine more closely those concentrations of MK92K approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was observed up to the highest five concentrations across all strains in the absence and presence of S-9.

Experiment 2 additional treatments of TA102 were performed in the absence of S-9 using a plate-incorporation methodology as insufficient concentrations were available to allow a thorough assessment of mutagenic activity. A narrowed concentration range was employed (1.95 ‑ 125 mg/plate). Following these treatments, evidence of toxicity was observed at 62.5 mg/plate and above.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

Following MK92K treatments of all the test strains in the absence and presence of S‑9, no increases in revertant numbers were observed that were concentration related, reproducible and statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any MK92K mutagenic activity in this assay system.

It was concluded that MK92K did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to at least 125 mg/plate (toxic concentration), in the absence and in the presence of a rat liver metabolic activation system (S‑9).