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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 January 1999 to 16 February 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UKEMS Guidelines
Version / remarks:
1990
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-isopropyl-9H-thioxanthen-9-one
EC Number:
226-827-9
EC Name:
2-isopropyl-9H-thioxanthen-9-one
Cas Number:
5495-84-1
Molecular formula:
C16H14OS
IUPAC Name:
2-(propan-2-yl)-9H-thioxanthen-9-one
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: yellow powder

Method

Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
- Five bacterial strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were used in this study.
- For all assays, bacteria were cultured for 10 hours at 37 °C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102). Incubation was carried out in a shaking incubator.
- Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics of histidine dependence, rfa character and resistance to ampicillin (TA98 and TA100) or ampicillin plus tetracycline (TA102). All experimentation commenced within 2 hours of the end of the incubation period
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
- Dose Range-finder Experiment and Mutation Experiment 1: 8, 40, 200, 1000 and 5000 µg/ plate
- Mutation Experiment 2: 51.2, 128, 320, 800, 2000 and 5000 µg/ plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test material solutions were prepared by dissolving the test material in anhydrous analytical grade dimethyl sulphoxide (DMSO), with the aid of vortex mixing, immediately prior to assay to give the maximum required treatment solution concentration. In Experiment 2, the formulation was warmed at 37 °C in order to achieve full dissolution of the test article. This solution was filter sterilised (Gelman Acrodisc CR filter, 0.2 μ.m pore size) and further dilutions were made using DMSO. The test material solutions were protected from light and used within approximately 5 hours of the initial formulation of the test material. All test material formulations and treatments were performed under subdued lighting.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene (5 µL/plate with metabolic activation) and Glutaraldehyde (25 µL/platefor TA102 without metabolic activation)
Remarks:
Controls were performed using the same addition volumes per plate as the test material treatments.
Details on test system and experimental conditions:
RANGE-FINDER EXPERIMENT
- The test material was tested for toxicity in strain TA100 in triplicate plates without and with S-9 mix. Negative (solvent) 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 °C:
0.1 mL bacterial culture,
0.1 mL test material solution or control and
0.5 mL 10 % S-9 mix or buffer solution
followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37 °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
- The test material was tested for mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments using triplicate plates without and with S-9. Experiment 1 mutagenicity data for strain TA 100 were provided by the range-finder experiment treatments. Negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S-9. Platings were achieved as described above.
- As the results of the first experiment were equivocal, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step, where the quantities of test material or control solution (reduced to 0.05 mL), bacteria and S-9 mix, were mixed together and incubated for 1 hour at 37 °C, with shaking, before the addition of 2.5 mL molten agar at 46 °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 solvent (DMSO) employed in this study. This, and some other organic solvents, 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 solvent that may have otherwise occurred.

COLONY COUNTING
- Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) or manually where confounding physical factors (such as split agar or the presence of test material precipitate) affected the accuracy of the automated counter. The background bacterial lawn of the plates was inspected for signs of toxicity.
Evaluation criteria:
ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
- the mean negative control counts fell within the normal ranges
- the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
- no more than 5 % of the plates were lost through contamination or some other unforeseen event.

EVALUATION CRITERIA
The test material was considered to be mutagenic if:
- the assay was valid
- Dunnett's test gave a significant response (p ≤ 0.01), and the data set(s) showed a significant dose-correlation
- the positive responses were reproducible.
Statistics:
- Individual plate counts from both experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined.
- The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TOXICITY, SOLUBILITY AND DOSE SELECTION
- An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of the test material at 8, 40, 200, 1000 and 5000 μg/plate, plus negative (solvent) and positive controls. No evidence of toxicity was observed following any of these treatments. Precipitation of the test material was observed on all plates treated at test material concentrations of 1000 and 5000 μg/plate, both in the absence and in the presence of S-9. These results were considered to be acceptable for mutagenic assessment and were used to provide the TA100 mutagenicity data for Experiment 1.
- In order to assess any potential biological effects (such as toxicity or mutagenicity) occurring over the precipitating doses in the remaining test strains, Experiment 1 treatments retained the dose-range employed in the range-finder experiment. Evidence of toxicity indicated by a reduction in revertant numbers was observed in strain TA 102 following treatments at 5000 μg/plate in the absence of S-9 and at 1000 μg/plate and above in the presence of S-9. No toxic signs were apparent following any treatments of the remaining test strains. Precipitation of the test article was again observed on plates treated at 1000 μg/plate and above.
- As some evidence of toxicity and mutagenicity was observed over the precipitating range in Experiment 1, Experiment 2 treatments of all the test strains retained 5000 μg/plate as the maximum test dose. A narrowed dose-range was employed in order to fully investigate those concentrations of test material considered most likely to induce any mutagenic effect. Furthermore the dose-range was extended in order to investigate both soluble and precipitating test doses. In addition the metabolic activation conditions were modified to include a pre-incubation step. Clear evidence of toxicity (in the form of a reduction in revertant numbers) was observed following treatments of strain TA98 at the maximum test dose in the presence of S-9 only and strain TA102 at test doses of 800 μg/plate and above, both in the absence and in the presence of S-9. Test material precipitate was observed on all plates treated at concentrations of 800 μg/plate and above.

MUTATION
- The individual plate counts were averaged to give mean values. From the data it can be seen that mean solvent control counts fell within the normal historical ranges and that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains which fell either within or above the normal historical ranges. Less than 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.
- The mutation data for the individual strains were evaluated as follows:
Test material treatments of strain TA98 in the absence of S-9, resulted in small but statistically significant increases in revertantnumbers, when the data were analysed at the 1 % level using Dunnett's test. These revertant number increases were reproduced over two independent experiments and demonstrated some evidence of a dose relationship (occurring at the maximum test dose in Experiment 1 and at the two highest test doses in Experiment 2). In both experiments the mutagenic response occurred at concentrations where test material precipitate was also evident. This pattern of mutagenic response within the precipitating range is deemed unusual. In addition the magnitude of revertant number increases observed was very low, and therefore it may be considered that the responses occurred at the limit of detection of this assay system. However, it was considered that overall these data provided some evidence of test material mutagenic activity, albeit weak.
- No test material treatments of any of the remaining strains or treatments of strain T A98 in the presence of S-9 resulted in any statistically significant increases in revertant numbers (following analysis of the data at the 1 % level using Dunnett's test).

Any other information on results incl. tables

Table 1: Summary of Experiment 1

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

 

TA100

TA1535

TA102

TA98

TA1537

-

Solvent

8

40

200

1000

5000

108

104

111

111

91*

92*

14

17

16

12

19*

15*

310

270

250

192

205*

124*

38

36

39

37

44*

54*

8

8

10

10

8*

11*

+

Solvent

8

40

200

1000

5000

123

123

105

104

106*

102*

16

14

12

15

17*

12*

269

251

216

180

116*

80*

45

47

37

40

42*

52*

7

8

7

9

9*

5*

Positive Controls

-

Name

SA

SA

GLU

2NF

9AA

Concentration (µg/plate)

2

2

25

5

50

Mean no. colonies/plate

655

488

712

972

487

+

Name

2AA

2AA

-

2AA

-

Concentration (µg/plate)

5

5

-

5

-

Mean no. colonies/plate

1966

214

 

2155

 

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

GLU = Glutaraldehyde

*= Precipitation

  

Table 2: Summary of Experiment 2

± S9 Mix

Concentration

(µg/plate)

Mean number of colonies/plate

 

TA100

TA1535

TA102

TA98

TA1537

-

Solvent

51.2

128

320

800

2000

5000

115

122

109

110

98*

98*

101*

17

17

17

13

15*

16*

13*

369

314

341

316

237*

221*

209*

38

36

46

40

42*

52*

68*

9

8

9

9

9*

7*

15*

+

Solvent

51.2

128

320

800

2000

5000

134

143

148

139

117*

109*

125*

19

15

21

20

15*

14*

15*

463

477

469

374

233*

204*

164*

41

34

38

32

32*

28*

21*

9

10

12

10

7*

6*

6*

Positive Controls

-

Name

SA

SA

GLU

2NF

9AA

Concentration (µg/plate)

2

2

25

5

50

Mean no. colonies/plate

689

458

786

970

634

+

Name

2AA

-

-

2AA

-

Concentration (µg/plate)

5

-

-

5

-

Mean no. colonies/plate

1809

-

-

1731

-

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

GLU = Glutaraldehyde

*= Precipitation

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, it was concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) did display evidence of weak mutagenic activity in Salmonella typhimurium strain TA98 when tested in the absence of metabolic activation, at precipitating test material doses (up to 5000 μg/plate).
Executive summary:

The potential of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B14 under GLP conditions.

The test material 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.

An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of the test material at 8, 40, 200, 1000 and 5000 μg/plate, plus negative (solvent) and positive controls. No evidence of toxicity was observed following any of these treatments. These results were considered to be acceptable for mutagenic assessment and were used to provide the TA100 mutagenicity data for Experiment 1.

Experiment 1 treatments retained the dose-range employed in the range-finder experiment. Evidence of toxicity indicated by a reduction in revertant numbers was observed in strain TA 102 only following treatments at the higher test doses in the absence of S-9 and in the presence of S-9.

Experiment 2 treatments of all the test strains retained 5000 μg/plate as the maximum test dose. A narrowed and extended dose-range was employed in order to fully investigate the mutagenic potential of both soluble and precipitating concentrations of the test article. In addition the metabolic activation conditions were modified to include a pre-incubation step. In this way it was hoped to increase the range of mutagenic chemicals that could be detected in this assay. Clear evidence of toxicity (in the form of a reduction in revenant numbers) was observed following treatments at the highest test doses in strain TA98 in the presence of S-9 and strain TA102 in the absence and in the presence of S-9.

Precipitation of the test material was observed on all plates treated at 800 μg/plate and above, both in the absence and in the presence of S-9.

Negative (solvent) 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.

Test material treatments of strain TA98 in the absence of S-9, resulted in small but statistically significant increases in revertant numbers, when the data were analysed at the 1 % level using Dunnett's test. These revertant number increases occurred at doses where test article precipitate was also evident. This weak mutagenic response was reproducible and demonstrated some evidence of a dose relationship.

Under the conditions of this study, it was concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) did display evidence of weak mutagenic activity in Salmonella typhimurium strain TA98 when tested in the absence of metabolic activation, at precipitating test material doses (up to 5000 μg/plate).