2,4-diethyl-9H-thioxanthen-9-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1) Four bacterial reverse mutation assays / Ames tests [OECD 471; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)]:

a) negative with and without activation in Salmonella typhimurium strains TA100, TA1535, TA1537 and TA102, negative with activation in TA 98 and positive without activation in TA98;

b) negative with and without activation in Salmonella typhimurium strains TA100 and TA1537, negative with activation in TA1535, negative with activation in TA 98 and positive without activation in TA98;

c) negative with and without activation in Salmonella typhimurium strains TA100, TA 1535 and TA1537, negative with activation in TA 98 and positive without activation in TA98;

negative with and without activation in Escherichia coli WP2uvrA;

d) negative with and without activation in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

2) In vitro mammalian chromosome aberration test [OECD 473; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)]: not clastogenic (negative with and without activation).

3) In vitro mammalian cell gene mutation test [OECD 476; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)]: positive (with and without activation).

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).

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.

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.

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).

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

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).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 May 2003 to 29 August 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

MEDIA USED
Oxoid nutrient broth #2, minimal glucose overlay containing histidine.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

MEDIA USED
Oxoid nutrient broth #2, minimal glucose overlay containing biotin/tryptophan.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- 61.7, 185.2, 555.6, 1666.7 and 5000.0 μg/plate.
- Precipitate appearead in the 1666.7 and 5000.0 μg/plate in the preliminary test, but it didn't interfere with the colony counting and was therefore used in the main test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test material was > 50 mg/ml in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitroquinoline-1-oxide (without metabolic activation) and 2-aminoanthracene (with metabolic activation).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: > 10^9/ mL
- The plate incorporation method was used. For the assay without metabolic activation, 0.1mL of the test material solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of sterile buffer were mixed with 2.0 mL of overlay agar; For the assay with metabolic activation, 0.1 mL of the test solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of S9-mix were mixed with 2.0 mL of overlay agar. After the contents of each tube was mixed, it was poured over the surface of the minimal agar plate. The overlay agar was allowed to solidify before incubation.
- All plates were incubated at 37 °C for 48 hours. After the incubation period, the number of revertant colonies in each plate was counted.

NUMBER OF REPLICATIONS: 3

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA100 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn’t interfere the colony counting.
- For TA98, the number of revertant colonies induced by ITX exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9 mix, and it demonstrated obvious concentration dependent relationship. Therefore, genotoxic effect of the test material was positive.

Table 1: Summary of Experiment

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 61.7 185.2 555.6 1666.7 5000.0	131 61* 36* 42* 48*† 93†	14 15 14 15 8*† 12†	35 35 35 38 34† 39†	33 31 37 47 77† 128†	15 8* 6* 8* 7*† 17†
+	Solvent 61.7 185.2 555.6 1666.7 5000.0	153 164 109 46* 65*† 94†	15 13 12 10 14† 14†	35 38 31 37 31† 26†	35 29 34 28 25† 29†	24 26 22 17* 19† 15†
Positive Controls
-	Name	4NQO	SA	4NQO	4NQO	9AA
	Concentration (µg/plate)	0.5	0.25	0.5	0.5	50
	Mean no. colonies/plate	1084	399	340	524	788
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	0.25	1.0	1.0	0.25	1.0
	Mean no. colonies/plate	1692	470	321	776	690

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

* = Inhibition

† = Precipitation

Conclusions:

Under the conditions of this study, the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was determined to be mutagenic to Salmonella typhimurium TA98 in the absence of metabolic activation.

Executive summary:

The mutagenic activity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471.

The test material was investigated in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA. The plate incorporation method was used in the assay and plates were tested either with or without metabolic activation in the form of S9 mix. All plates were incubated at 37 °C for 48 hours. After the incubation period, the number of revertant colonies in each plate was counted.

The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn’t interfere the colony counting. The test material was prepared in DMSO.

For TA98, the number of revertant colonies induced by the test material exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9 mix, and it demonstrated obvious concentration dependent relationship. The test material also showed inhibitory effects on Salmonella typhimurium: TA100 and TA1537 with and without metabolic activation and TA1535 without metabolic activation.

Under the conditions of this study, the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was determined to be mutagenic to Salmonella typhimurium TA98 in the absence of metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 May 2003 to 29 August 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Media Used: OXOID Nutrient broth#2, Minimal glucose agar, overlay agar containing histidine and biotin/tryptophan.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Media Used: OXOID Nutrient broth#2, Minimal glucose agar, overlay agar containing histidine and biotin/tryptophan.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Doses: 61.7, 185.2, 555.6, 1666.7, 5000.0 µg/plate with or without metabolic activation.
- Rationale for Dosage Selection: The precipitate appeared in 1666.7-5000.0 µg/plate in preliminary test, but it didn't interfere with the colony counting.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test material was > 50 mg/mL in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

- The plate incorporation method was used.
- For the assay without metabolic activation, 0.1 mL of the test solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of sterile buffer were mixed with 2.0 mL of overlay agar; For the assay with metabolic activation, 0.1 mL of the test solutions, 0.1 mL of fresh bacterial culture and 0.5 mL of S9-mix were mixed with 2.0 mL of overlay agar.
- After the contents of each tube was mixed, it was poured over the surface of the minimal agar plate.
- The overlay agar was allowed to solidify before incubation.
- Incubation: All plates were incubated at 37 °C for 48 hours. After the incubation period, the number of revertant colonies in each plate was counted.
- Three plates were used per concentration.
- Number of cells per culture: > 10^9/mL

Evaluation criteria:

After the incubation period, the number of revertant colonies in each plate was counted.

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 applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 100, TA 1535 & TA 1537

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

- The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn't interfere the colony counting.
- For TA98, the number of revertant colonies induced by the test material exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9-mix, and it demonstrated obvious concentration dependent relationship.
- Therefore, the genotoxic effect of the test material was positive.

Table 1: Summary of Results

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 61.7 185.2 555.6 1666.7 5000.0	133 61† 36 † 42 † 48* † 93*	14 15 14 15 8*† 12*	35 35 35 38 34* 39*	33 31 37 47 77* 128*	15 8† 6† 8† 7*† 17*
+	Solvent 61.7 185.2 555.6 1666.7 5000.0	153 164 109 46 † 65*† 94*	15 13 12 10 14* 14*	35 38 31 37 31* 26*	35 29 34 28 25* 29*	24 26 22 17† 19* 15*
Positive Controls
-	Mean no. colonies/plate	1084	399	340	524	788
+	Mean no. colonies/plate	1692	470	321	776	690

* = Precipitation

† = Inhibition

Conclusions:

Under the conditions of this study the genotoxic effect of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was positive to Salmonella typhimurium TA 98 without metabolic activation. The test material was negative to TA 98 with metabolic activation and to all other tested strains with or without metabolic activation.

Executive summary:

The genetic toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was investigated in accordance with the standardised guideline OECD 471.

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2urvA, were subjected to the test material using the plate incorporation method. Doses of 61.7, 185.2, 555.6, 1666.7, 5000.0 µg/plate were tested with or without metabolic activation, in the form of S9-mix. All plates were incubated at 37 °C for 48 hours. After the incubation period, the number of revertant colonies in each plate was counted.

The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn't interfere the colony counting. For TA98, the number of revertant colonies induced by the test material exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9-mix, and it demonstrated obvious concentration dependent relationship.

Under the conditions of this study the genotoxic effect of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was positive to Salmonella typhimurium TA 98 without metabolic activation. The test material was negative to TA 98 with metabolic activation and to all other tested strains with or without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 July 1987 to 20 July 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- The strains are tested routinely for cell membrane permeability and where applicable for ampicillin resistance.
- For use in tests sub-cultures are grown in Nutrient Broth (Oxoid) at 37 °C for 18 hours . This culture provides approximately 2 x 10^9 organisms per mL which is assessed by cell counting.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Dose range finding test: 5, 50, 500 and 5000 µg/ plate. The test material was not toxic towards the tester strains. Therefore 5000 μg/plate was chosen as the top dose level in the mutation tests.
- Mutation test: 50, 150, 500, 1500 and 5000 µg/ plate

Vehicle / solvent:

- Vehicle(s)/solvent used: Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene 2 µg/plate for TA 1535 and TA 1537 with S9 and 0.5 µg/platefor TA 98 and TA 100 with S9

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST
- Four concentrations of test material are assessed for toxicity using the four tester strains. The highest concentration is usually 0.05 g of test material dissolved in 1 mL of solvent. Three 10-fold serial dilutions of the top concentration are also tested. The chosen solvent is used as the negative control.
- 0.1 mL of an overnight bacterial culture containing approximately 2 x 10^9 cells/mL, and 0 .5 ml S-9 mix or 0.5 mL 0.1 M sodium phosphate buffer (pH 7.4) are placed in glass bijou bottles. 0.1 mL of the test solution is added followed by 2 mL histidine deficient agar. The mixture is thoroughly shaken and overlaid onto previously prepared plates containing 20 mL minimal agar. Single petri dishes are used for each dose level. They are incubated at 37 °C for 72 hours . After this period the plates are examined for the appearance of a complete bacterial lawn. Revertant colonies are counted using a Biotran Automatic Colony Counter. Any toxic effects of the test material are detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

MAIN TEST PROCEDURE
- Without metabolic activation
0 .1 mL aliquots of bacterial suspension and 0.5 mL of sterile 0.1 M sodium phosphate buffer (pH 7.4) are added to each of one set of sterile bijou bottles.
0.1 mL of the test material is added to cultures at five concentrations separated by half-log 10 intervals . The negative control is the chosen solvent. The appropriate positive control is also included. 3 bottles are used at each dose level .
2.0 mL of histidine deficient agar is added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 20 mL of minimal agar. Plates are incubated for 72 hours at 37 °C.
Colonies are counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.
- With metabolic activation
Methodology is as described in the procedure ‘without metabolic activation’ except that 0.5 mL of liver homogenate S-9 mix is added to bijou bottles in place of sterile buffer.
- Second Mutation Test
The procedure outlined is repeated at a later date; though the concentrations of test material used in the second test may be altered, if the results of the first test indicate this may be expedient.

Evaluation criteria:

ASSESSMENT OF RESULTS
- The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups.
- The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
- The test material is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments and the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 examined

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 examined

Positive controls validity:

valid

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 examined

Positive controls validity:

valid

Additional information on results:

- The test material was not toxic towards the tester strains. Therefore 5000 μg/plate was chosen as the top dose level in the mutation tests.
- No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test material at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Conclusions:

Under the conditions of the study, it is concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not mutagenic in the Salmonella typhimurium reverse mutation assay.

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 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100). The test was performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

A preliminary dose range finding test was performed at 5, 50, 500 and 5000 µg/ plate. The test material was not toxic towards the tester strains. Therefore 5000 μg/plate was chosen as the top dose level in the mutation tests.

The main test was performed at 50, 150, 500, 1500 and 5000 µg/ plate both with and without metabolic activation (S9 mix). Agar plates were incubated for 72 hours at 37 °C. Colonies were counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.

The test material was not toxic towards the tester strains. No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test material at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Under the conditions of the study, it is concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 June 2003 to 15 October 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver microsomal fraction

Test concentrations with justification for top dose:

Test 1 (With activation): 37.5, 70 and 150 µg/mL
Test 1 (Without activation): 15, 30 and 60 µg/mL
Test 2 (Without activation): 10, 20 and 40 µg/mL
Test 3 (Without activation): 2.5, 5 and 10 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

WITH METABOLIC ACTIVATION
Test 1- Exposure time: 6 hours and Harvest time: 24 hours

WITHOUT METABOLIC ACTIVATION
Test 1- Exposure time: 6 hours and Harvest time: 24 hours

WITHOUT METABOLIC ACTIVATION
Test 2- Exposure time: 24 hours and Harvest time: 24 hours

WITHOUT METABOLIC ACTIVATION
Test 3- Exposure time: 48 hours and Harvest time: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was determined with or without metabolic activation using viable cell counts.

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In the cytotoxicity measurement, it was determined that concentrations of the test material at 60 µg/mL, 40 µg/mL and 10 µg/ml respectively for 6 hours, 24 hours and 48 hours without metabolic activator and at 150 µg/mL with metabolic activator could result in a significant inhibition of cell growth by 50 percent.
- Without metabolic activator, the test material did not cause structural chromosome aberration in CHL cells exposed for 6 hours, 24 hours and 48 hours respectively, but it could induce the increase of gap in CHL cells treated for 48 hour. The test material didn't induce structural chromosome aberration in CHL cells with metabolic activator.
- The test material caused a significant increase of polyploidy in CHL cells at 60 µg/mL in the case of CHL cells exposed for 6 hours without metabolic activation (P<0.05). No other abnormality was observed.

- In this study, it was observed that without metabolic activation, polyploidy of CHL cells increased significantly exposed for 6 hours at 60 µg/mL of test material, while this case didn't occur when exposed for 24 hours and 48 hours, respectively. Since the polyploidy ratio is as low as 2.5 % and there was no obvious dose-response relationship, it is therefore concluded that the 6 hours result may be not related to action of the test material.

Table 1: Effect of the Test Material on Structural Chromosome Aberration in CHL Cells

Metabolic activation	Exposure period	Dose (µg/mL)	Cell no. scored	Gaps	No. of structural aberrations	Ratio of aberration (%)	Result
+	6 hours	0	200	3	2	1.0	-
		37.5	200	6	3	1.5	-
		75	200	12	1	0.5	-
		150	200	8	8	4.0	-
		CP (20)	200	28	118	59.0	+
-	6 hours	0	200	0	3	1.5	-
		15	200	6	3	1.5	-
		30	200	2	1	0.5	-
		60	200	3	6	3.0	-
		MMC (0.2)	200	5	73	36.5	+
-	24 hours	0	200	3	3	1.5	-
		10	200	2	4	2.0	-
		20	200	5	2	1.0	-
		40	200	2	2	1.0	-
		MMC (0.1)	200	30	106	53.0	+
-	48 hours	0	200	0	2	1.0	-
		2.5	200	14	2	1.0	-
		5	200	10	7	3.5	-
		10	200	18	8	4.0	-
		MMC (0.05)	200	4	41	20.5	+

 

Table 2: Effect of the Test Material on Polyploidy in CHL Cells

Metabolic activation	Exposure period	Dose (µg/mL)	Cell no. scored	Number of polyploid	Ratio of polyploidy (%)	X^2
+	6 hours	0	200	4	2.0	5.44 P > 0.05
		37.5	200	5	2.5
		75	200	2	1.0
		150	200	0	0.0
		CP (20)	200	0	0.0
-	6 hours	0	200	0	0.0	9.09 P > 0.05
		15	200	3	1.5
		30	200	0	0.0
		60	200	5	2.5*
		MMC (0.2)	200	0	0.0
-	24 hours	0	200	2	1.0	2.74 P > 0.05
		10	200	3	1.5
		20	200	2	1.0
		40	200	0	0.0
		MMC (0.1)	200	0	0.0
-	48 hours	0	200	0	0.0	3.0 P > 0.05
		2.5	200	1	0.5
		5	200	0	0.0
		10	200	0	0.0
		MMC (0.05)	200	0	0.0

Compared with Negative Control:*, P<0.05

Conclusions:

Under the conditions of this study, the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not clastogenic in either the presence or absence of metabolic activation.

Executive summary:

The potential of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) to cause chromosome aberrations in cultured mammalian cells was investigated in accordance with the standardised guideline OECD 473.

CHL/IU cells were treated with the test material according to the following:

- Test 1 (with metabolic activation) for exposure time 6 hours and harvest time 24 hours at 37.5, 70 and 150 µg/mL;

- Test 1 (without metabolic activation) for exposure time 6 hours and harvest time 24 hours at 15, 30 and 60 µg/mL;

- Test 2 (without metabolic activation) for exposure time of 24 hours and harvest time 24 hours; and

- Test 3 (without metabolic activation) for exposure time 48 hours and harvest time 48 hours.

Metabolic activation was provided in the form of Induced rat liver microsomal fraction. The test material was prepared in DMSO and this was used as the solvent control. Mitomycin C was used as a positive control in the absence of exogenous metabolic activation and Cyclophosphamide was used for the presence of exogenous metabolic activation. Cytotoxicity was determined with or without metabolic activation using viable cell counts.

In the cytotoxicity measurement, it was determined that concentrations of the test material at 60 µg/mL, 40 µg/mL and 10 µg/mL respectively for 6 hours, 24 hours and 48 hours without metabolic activation and at 150 µg/mL with metabolic activator could result in a significant inhibition of cell growth by 50 percent.

Without metabolic activation, the test material did not cause structural chromosome aberration in CHL cells exposed for 6 hours, 24 hours and 48 hours respectively, but it could induce the increase of gaps in CHL cells treated for 48 hours. The test material didn't induce structural chromosome aberration in CHL cells with metabolic activator.

The test material caused a significant increase of polyploidy in CHL cells at 60 µg/mL in the case of CHL cells exposed for 6 hours without metabolic activation (P<0.05). No other abnormality was observed.

In this study, it was observed that without metabolic activation, polyploidy of CHL cells increased significantly when exposed for 6 hours at 60 µg/ml of test material, while this case didn't occur when exposed for 24 hours and 48 hours, respectively. Since the polyploidy ratio is as low as 2.5 % and there was no obvious dose-response relationship it is therefore concluded that the 6 hours results may not be related to action of the test material.

Under the conditions of this study, the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not clastogenic in either the presence or absence of metabolic activation.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian cell gene mutation test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Cells are frozen in liquefied nitrogen. Cells are propagated from stock cultured, seeded in the RPMl1640 culture medium (10 % equine serum) and incubated at 37 °C, 5% CO2.
- Number of Cells passages <10 passages

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver microsomal fraction (S9)

Test concentrations with justification for top dose:

20, 30, 45, 67.5 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

- Composition of media (R10): 89 % RPMI1640, 10 % Equine serum, 1 % Benzylpenicilin-Streptomycin and 200 µg/mL sodium pyruvate.
 
- Test conditions:
-Incubation temperature: 37 °C
-CO2 concentration: 5 %
-Duration of treatment: 3 hours
-Cell density during treatment: 5 x 10^5/mL
 
- Induced rat liver microsomal fraction (S9) ingredients: Glucose-6-phosphate 180 mg/mL, NADP 25 mg/mL, KCl 150 mM and Rat liver S9.
 
- Expression period: The expression period was 48 hours. The number of cells seeded was 2 x 10^5/mL in the first day when the survival plate (PEO) is done. The cell density was adjusted to 2 x 10/mL on the next day. On the third day cells were harvested plated on the visibility plate (PE2) and mutant frequency plate (MF).
 
- Selective agent: Trifluorothymine deoxyribose (3 µg/mL)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The conclusion of the test was valid since the results of PEO, PE2, MF and SC of the negative and positive control were in normal range.
- It was observed the mutant frequency increased at 20.0, 30.0 and 45.0 µg/mL. The outcome can't be observed in 67.5 µg/mL group without metabolic activation because 67.5 µg/mL was too high so the cells at that concentration were dead. There was a concentration-related increase in mutant frequency from 20.0 to 45.0 µg/mL without and with metabolic activation, and the scores of small clone ascended with the concentrations in the treatment groups.

Table 1: Result of Mammalian Gene Mutation Test Without Metabolic Activation

Test material	Concentration (µg/mL)	PEO	RSO (%)	PE2	RS2	MF (x10^-6)	SC (%)
Negative control	0.0	1.01	100.0	1.16	100.0	121.8	30.1
I	20.0	0.48	47.4	0.93	79.8	227.5*	36.7
II	30.0	0.34	33.4	0.86	74.5	314.6*	47.3
III	45.0	0.23	22.8	0.34	29.2	422.2*	71.2
IV	67.5	0.01	0.6	-	-	-	-
Positive control (MMS)	10.0	1.27	125.0	0.82	70.4	617.6*	56.3

PE: Plate Efficiency; MF: Mutant Frequency; SC: Small Clone; RS: Relative Survival

Under the conditions of this study the test material was observed to be positive in the Mammalian Gene Mutation Test.

 

Table 2: Result of Mammalian Gene Mutation Test With Metabolic Activation

Test material	Concentration (µg/mL)	PEO	RSO (%)	PE2	RS2	MF (x10^-6)	SC (%)
Negative control	0.0	0.84	100.0	0.98	100.0	192.7	32.9
I	20.0	0.70	83.5	0.69	70.1	373.9*	36.5
II	30.0	0.50	60.0	0.74	75.6	374.5*	43.7
III	45.0	0.23	28.0	0.75	76.6	994.4*	56.3
IV	67.5	0.12	14.2	0.98	99.7	514.5*	60.1
Positive control (CP)	2.0	0.75	89.4	1.06	108.7	426.5*	48.4
Positive control (CP)	3.0	0.64	76.0	0.92	94.1	638.2*	52.6

PE: Plate Efficiency; MF: Mutant Frequency; SC: Small Clone; RS: Relative Survival

 

Table 3: Dose-Response Relationship of the Test Material Without and With Metabolic

Activation

Metabolic activation	B^a
Without	6.01E^-6**
With	6.22E^-6**

A = B is the slope

** P < 0.01

Conclusions:

Under the conditions of this study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was observed to be positive in the Mammalian Gene Mutation Test.

Executive summary:

The genetic toxicity of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was investigated in the mammalian gene mutation test in accordance with the standardised guideline OECD 476.

L5178Y mouse lymphoblastoid cells were treated with the test material for 48 hours at the following concentrations: 20.0, 30.0, 45.0 and 67.5 µg/mL. Cells were treated both in the presence and absence of metabolic activation in the form of induced rat liver microsomal fraction (S9). Trifluorothymine deoxyribose (3 µg/mL) was used as the selective agent.

The conclusion of the test was valid since the results of PEO, PE2, MF and SC of the negative and positive control were in normal range.

It was observed the mutant frequency increased at 20.0, 30.0 and 45.0 µg/mL. The outcome can't be observed in 67.5 µg/mL group without metabolic activation because 67.5 µg/mL was too high so the cells at that concentration were dead. There was a concentration-related increase in mutant frequency from 20.0 to 45.0 µg/mL without and with metabolic activation, and the scores of small clone ascended with the concentrations in the treatment groups.

Under the conditions of this study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was observed to be positive in the Mammalian Gene Mutation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

1) In Vivo Mammalian Erythrocyte Micronucleus Test [OECD 474; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)]: negative (not mutagenic).

2) Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo [OECD 486; test material: 2-Isopropylthioxanthone (CAS 5495-84 -1, EC 226-827-9)]:

negative (not genotoxic to male Wistar rats).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2004 to 09 August 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Species:

mouse

Strain:

NMRI

Remarks:

BR

Details on species / strain selection:

Animals are recommended by international guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 29.4 to 31.2 g. The bodyweights at the start of the treatment were within 20 % of the sex mean.
- Fasting period before study: Feed was withheld 3 to 5 h prior to dosing until administration.
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding. Paper bedding was provided as nest material.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30 to 70 %
- Air changes: 15 air changes per hour.
- Photoperiod: The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Propylene glycol
- Amount of vehicle: The dosing volume was 10 mL/kg bodyweight.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test material concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Test material concentrations were dosed within 2 hours after preparation.

Duration of treatment / exposure:

A single dose via gavage

Frequency of treatment:

Once

Post exposure period:

24 and 48 hours

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Three animals per sex per dose in the dose range-finding study.
Five males per dose in the main test.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- Route of administration: Oral
- Vehicle: Physiological saline
- Doses / concentrations: 50 mg/kg body weight

Tissues and cell types examined:

All slides were randomly coded before examination. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Details of tissue and slide preparation:

ISOLATION OF BONE MARROW
Bone marrow of the groups treated with the test material was sampled 24 or 48 hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 mL of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm ( approximately 100 g) for 5 min.

PREPARATION OF BONE MARROW SMEARS
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1 :1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue) and marked. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.

STAINING OF THE BONE MARROW SMEARS
The slides were automatically stained using the 'Wright-stain-procedure" in an "Ames" HEMAtek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean± three times the standard deviation): Males: 1.3 ‰ ± 3.9 ‰ indicated are means for n=245.

Statistics:

- A test material is considered positive in the micronucleus test if it induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
- A test material is considered negative in the micronucleus test if none of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
-The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE-FINDING STUDY
- In a dose range finding study 6 animals (3 males and 3 females) were dosed orally with 2000 mg/kg body weight of test material. The animals showed no abnormalities after dosing during three days.
Based on the results of the dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus main test.

MAIN STUDY
- Mortality and systemic toxic signs: All animals of the groups treated with the test material and the animals of the negative and positive control group showed no abnormalities.

- Micronucleated polychromatic erythrocytes: No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

- Ratio polychromatic to normochromatic erythrocytes: The animals of the groups which were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Conclusions:

Under the conditions of this study it is concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not mutagenic in the micronucleus test.

Executive summary:

The genetic toxicity in vivo of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was investigated in a study performed in accordance with the standardised guidelines OECD 474 and EU Method B.12 under GLP conditions.

The test material was investigated in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The test material was formulated in propylene glycol.

Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 2000 (two groups), 1000 (one group), and 500 (one group) mg/kg body weight. The animals of all dose levels showed no abnormalities after dosing.

Bone marrow of the groups treated with the test material was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control group was harvested 24 and 48 hours after dosing, respectively. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test material.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of the test material on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

Under the conditions of this study it is concluded that the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) is not mutagenic in the micronucleus test.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2004 to 09 September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 8 weeks old.
- Weight at study initiation: 246 ± 11 g, range 228 - 273 g.
- Assigned to test groups randomly: Yes
- Fasting period before study: For the animals of the 12 to 16 hours sampling time, the feed was withheld 4 hours prior to dosing (late in the afternoon) until perfusion. The animals had free access to tap water. For the animals of the 2 to 4 hours sampling time, a limited quantity of food was supplied during the night before dosing (approximately 7 g/rat). The animals had free access to tap water. This feeding regimen is chosen to facilitate the process of perfusion.
- Housing: Animals were housed in groups of 3 animals per cage in labeled polycarbonate cages containing purified sawdust as bedding material. Cages were changed once a week. After exposure, animals were housed in individual treatment groups comprising 1 to 3 animals per group.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 %
- Air changes: Approximately 15 per hour.
- Photoperiod: The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was suspended in propylene glycol. In the dose range finding study formulations were blended and treated with ultrasonic waves to obtain a homogeneous suspension. In the main study a homogeneous suspension of the formulations was obtained by blending only. Test material concentrations were dosed within 45 minutes after preparation.

Duration of treatment / exposure:

The animals were treated once.

Frequency of treatment:

Once

Post exposure period:

2 to 4 hours and 12 to 16 hours

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 animals per sex per dose, per sampling time.

Control animals:

yes, concurrent vehicle

Positive control(s):

- 2 to 4 hour treatment time: Dimethylnitrosamine, 10 mg/kg body weight administered in water.
- 12 to 16 hour treatment time: 2-acetylaminofluorene, 50 mg/kg body weight administered in corn oil.

Tissues and cell types examined:

- The viability and number of hepatocytes were determined.
- The slides were checked for the presence of sufficient cells of normal morphology to permit a meaningful assessment of UDS
- Preparations were examined microscopically for signs of overt cytotoxicity (e.g. pyknosis).
- Slide evaluation was performed without knowledge of the treatment schedule.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose for the in vivo rat hepatocyte DNA-repair assay was based on a pilot study. One dose group, comprising 5 males, received a single dose of 2000 mg test material per kg body weight. The study duration was 2 days. During this period mortality and physical condition were recorded. The test substance showed no toxicity in the pilot study at 2000 mg/kg body weight, the highest dose accepted in the guidelines. Therefore, 2000 mg/kg was selected as the highest dose and 1000 mg/kg was selected as the lower dose to be tested.

IN VIVO RAT HEPATOCYTE DNA-REPAIR ASSAY
At least three male rats were used per sampling time in each treatment group, except for the vehicle and positive control group of which two animals were used per sampling time. The animals were dosed once and sampled at either 2 to 4 hours or 12 to 16 hours.

LIVER PERFUSION
- Rats were anaesthetised by intraperitoneal injection with Dormitor and Ketamine, both 0.3 mL/rat. The abdominal cavity was opened and the portal vein was ligated loosely to fix a catheter. Subsequently, a cannula (which was flushed with heparin) was inserted into the portal vein and the ligature was fixed. A heparin solution (0.2 mL) was injected into the spleen to prevent blood coagulation. The cannula was then connected to a reservoir with isolation media (Hanks' Balanced Salt Solution, Ca2 + and Mg2 + free, 37 °C), which was infused by a peristaltic pump, and immediately after this the inferior vena cava and the hepatic artery were cut. The pump rate was approximately 50 mL/minute. Approximately 400 mL HBSS was used to perfuse the liver. The liver was excised during perfusion with HBSS and placed in a glass beaker. Subsequently, a collagenase solution (37 °C, 0.05 % w/v Collagenase type IV for hepatocyte isolation) supplemented with 2.5x10^-3 mol.l^-1 CaCl2 was recirculated for ca. 10 minutes. The liver was transferred to a sterile petri dish and hepatocytes were isolated in a flow cabinet by cutting the Glisson capsules and shaking the liver in HBSS supplemented with 2.5x10^-3 mol.l^-1 CaCl2 and 2.5 % (w/v) Bovine Serum Albumin. In this way the tissue was dissociated into single cells.
- The obtained cell suspension was incubated at 37 °C under 95 ± 0.5 % O2 and 5 ± 0.5 % CO2, in a humid atmosphere for 10 minutes. Dead cells, endothelial cells and Kupffer cells were removed by centrifugation (50 g), whereas living parenchymic liver cells, the hepatocytes, were retained.
- Primary cell culture conditions: Viability of the hepatocytes was determined using the trypan blue dye exclusion method. After isolation, the hepatocytes were counted by microscope with an "Improved Neubauer" haemocytometer (Merck, the Netherlands) and seeded on 1 cm^2 cover slips in wells of a 24-well dish. Per cover slip 10^5 cells were seeded. Four cover slips were prepared per animal.
- Culture medium consisted of Williams E Medium supplemented with foetal calf serum (10 %), L-glutamine (2 x 10^-3 mol-1) and gentamycin (20 µL).
- All incubations were carried out in a humid atmosphere (80 - 100 % containing 5 % CO2 in air in the dark at 37 ± 1 °C. Temperature and CO2 percentage were monitored during the experiment.

LABELLING OF THE CELL CULTURES: The cultures were washed once with culture medium to remove unattached cells and debris 1 to 2 hours after seeding of the cells. Subsequently, 0.5 mL of culture medium supplemented with tritiated thymidine solution (3H-TdR; 370 KBq/mL (10 μCi/mL); specific activity 666 to 1110 GBq/mol (18 to 30 x 10^3 Ci/mol)) was added to the cells. The cultures were incubated for 4 hours at 37 °C (under 95 % O2 and 5 % CO2, in a humid atmosphere). After incubation the cultures were washed once with culture medium and 1.0 ml of culture medium containing 0.25x10^-3 M unlabelled thymidine solution was added to each well to diminish unincorporated radioactivity. Cultures were incubated overnight (14 to 18 hours).Thereafter the cultures were washed with HBSS and fixed with methanol-acetic acid (3:1 v/v).

AUTORADIOGRAPHIC PROCEDURE: After fixation of the cells the coverslips were mounted on microscopic slides. These slides were dipped in llford K5D emulsion (Polysciences) at 40 to 45 °C and dried for 2 hours at room temperature in the dark. After drying, the slides were placed in light tight boxes in the presence of silica gel. The photographic emulsion was exposed for 7 days at 4 °C. The emulsion was developed for 4 min in Kodak D19 developer at 15 °C, rinsed in Milli-RO water and fixed for 5 min in Kodak fixative. The slides were rinsed with running tap water and the cells were stained with haematoxylin/eosin. At least 3 scorable coverslips per animal were subjected to the autoradiographic procedure.

SCORING
- The slides were checked for the presence of sufficient cells of normal morphology to permit a meaningful assessment of UDS. Preparations were examined microscopically for signs of overt cytotoxicity (e.g. pyknosis). Slide evaluation was performed without knowledge of treatment schedule.
- Grain counts were determined over the nuclei (nuclear grains, NG) and the nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, CG) manually on the coverslips. In general, 50 cells were counted on each coverslip, and at least 2 coverslips per animal (in total 100 cells per animal) and 3 animals per datapoint were examined (except for the negative and positive controls where at least 1 animal was examined).
The following criteria were used to determine if a cell was countable:
- Cells with abnormal morphology, such as those with pyknotic or lysed nuclei, were not counted.
- Isolated nuclei not surrounded by cytoplasm were not counted.
- Cells with unusual staining artifacts or in the presence of debris were not counted.
- Heavily labelled cells in S-phase were not counted.
The normal cells observed while moving the microscope stage were counted.
- Averages and standard deviations were calculated. Grain counts over nuclear areas (NG) were compared to grain counts over the most heavily labelled adjacent cytoplasm area (CG) of the same size as the corresponding nuclear area. The net nuclear grain count (NNG) was calculated for each cell by subtracting CG counts from NG counts. The background counts of a single area of the same size as the corresponding nuclear area was recorded per coverslip.

Evaluation criteria:

DATA EVALUATION
- A test material is considered positive in the DNA-repair assay if it yields greater than or equal to 5 net nuclear grain (NNG) counts per group average, and greater than or equal to 20 % of the cells responding (i.e. with NNG values of 5 or more). These values are based on our laboratory historical data.
- A test material is considered negative in the DNA-repair assay if it does not induce a net nuclear grain (NNG) count ≥ 5 and ≥ 20 % of the cells responding.
In addition to the above-mentioned criteria, biological relevance of the data will also beconsidered, i.e. parameters such as inter-animal variation, dose response relationship and cytotoxicity will be taken into account.
- The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

ACCEPTABILITY OF THE ASSAYA
DNA-repair assay is considered acceptable if it meets the following criteria:
- The viability of the hepatocytes from the negative control animals should be at least 50 percent.
- The positive control substances should produce increases in the number of net nuclear grain counts, yielding greater than or equal to 5 net nuclear grain counts per group average, and greater than or equal to 20 % of the cells responding.
- The net nuclear grain count found in the negative controls should reasonably be within the laboratory historical control data range.
- The selected dose range should include a toxic dose as demonstrated by the preliminary dose range finding test. In case of a non-toxic test substance the dose range should extend to 2000 mg/kg body weight or to the limit of solubility.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE-FINDING STUDY
- In a pilot study at first one dose group (3 male rats) were dosed orally with 2000 mg/kg body weight. The animals did not show any signs of reaction to treatment.
- Two days after exposure a pilot perfusion was performed in two of the animals to recognise any adverse effects on the liver at this dose level that could interfere with a good performance of the main experiment. The liver was rigid, the tissue was not dissociated into single cells and the viability could not be determined. This was most likely caused by the procedure of the hepatocyte isolation, and was not test material related.
- Additionally two dose groups were dosed orally with 2000 (two males) and 0 (one male) mg/kg body weight. The animals did not show any signs of reaction to treatment. The vehicle control animal was added to compare possible problems during the liver perfusion between the different animal groups.
- Two days after exposure a pilot perfusion was performed in all animals to recognise any adverse effects on the liver that could interfere with a good performance of the main experiment. No macroscopic effects in the liver were observed and the viability of the liver cells was 73 and 60 % in both animals treated with 2000 mg/kg body weight, indicating that hepatotoxicity would not adversely influence the performance of the in vivo rat hepatocyte DNArepair assay. The viability of the liver cells of the vehicle control animal was 76 %.
- Based on the results of this pilot study and the pilot perfusion 2000 and 1000 mg /kg body weight were selected as appropriate doses for the in vivo rat hepatocyte DNA-repair assay.

IN VIVO RAT HEPATOCYTE DNA-REPAIR ASSAY
- Mortality/ signs of toxicity: All animals orally dosed with the test material survived the sampling period. All animals showed no abnormalities after dosing, except for one vehicle control animal, which was lethargic and had a hunched posture just prior to the perfusion.
- Viability of the hepatocytes: The viability of the hepatocytes, used for the evaluation of DNA repair inducing ability of the test material, was at least 73 %, indicating no direct liver toxicity. A viability of at least 77 % was found for the vehicle control cultures.
- Results DNA repair assay: In all slides used for grain counts sufficient cells of normal morphology to permit a meaningful assessment of unscheduled DNA-synthesis were present. Preparations showed no or slight overt cytotoxicity (e.g. pyknosis ≤ 50 %). As a result of oral dosing with the test material the NNG per coverslip and per animal, as well as the group average revealed no positive response in this assay at any of the dose levels. The percentage of cells in repair (repair taken as NNG ≥ 5), both per individual animal and for the group average, revealed no increase at any dose.
- Acceptability: The NNG in the solvent-treated control cultures was within the historical control data range. Oral dosing of a male rat with dimethylnitrosamine (DMN) resulted in a NNG of 37 with 100 % of the cells in repair (NNG ≥ 5). Oral dosing of a male rat with 2-acetylaminofluorene (2-AAF) resulted in a net nuclear grain count (NNG) of 27 with 99 % of the cells in repair (NNG ≥ 5). In the scored coverslips the mean background of a single area of the same size as the corresponding nuclear area, located outside the cytoplasm, was always less than 7 grains, indicating that the autoradiographic procedure functioned adequately. It can be concluded that the test system was functioning correctly.

Conclusions:

Under the conditions of this study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was not genotoxic to male Wistar rats.

Executive summary:

The DNA repair inducing ability of the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) in male Wistar rat hepatocytes, measured as unscheduled DNA synthesis (UDS) was investigated in accordance with the standardised guidelines OECD 486 and EU Method B39, under GLP conditions.

The test material was prepared in propylene glycol. Dose groups received a single oral dose of the test material. Hepatocytes were sampled 2-4 or 12-16 hours after dosing with 2000 and 1000 mg/kg body weight (b.w.). All animals showed no abnormalities after dosing, except for one vehicle control animal, which was lethargic and had a hunched posture just prior to the perfusion.

Corresponding vehicle treated groups served as negative controls (vehicle was propylene glycol). Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg b.w.) or 2-acetylaminofluorene (2-AAF, 50 mg/kg b.w.) were harvested 2-4 or 12-16 hours after dosing respectively. Two slides per animal and three animals per data point were examined.

As a result of oral dosing with the test material the net nuclear grain count (NNG) per coverslip and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG≥5), both per individual animal and for the group average, revealed no significant increase at any dose.

The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system was functioning correctly.

Under the conditions of this study the test material 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9) was not genotoxic to male Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No genetic toxicity studies are available for the registration substance 2,4-diethyl-9H-thioxanthen-9-one ("DETX", CAS 82799-44-8, EC 280-041-0) itself. However, endpoint information of the structurally related substance 2-Isopropylthioxanthone ("ITX", CAS 5495-84-1, EC 226-827-9) can be used for the assessment of the genotoxic potential of the substance DETX. Such a proceeding is considered entirely appropriate because the only difference between the source substance ITX and the target substance DETX can be reduced to slightly differing substituents: ITX possesses one isopropyl group, whereas DETX two ethyl groups.

Genetic toxicity in vitro

The mutagenic activity of the test material ITX was evaluated in a bacterial reverse mutation assay conducted in accordance with the standardised guideline OECD 471. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was investigated in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA. The plate incorporation method was used in the assay and plates were tested either with or without metabolic activation in the form of S9 mix. All plates were incubated at 37 °C for 48 hours. After the incubation period, the number of revertant colonies in each plate was counted.

The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn’t interfere the colony counting. The test material was prepared in DMSO.

For TA98, the number of revertant colonies induced by the test material exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9mix, and it demonstrated obvious concentration dependent relationship. The test material also showed inhibitory effects on Salmonella typhimurium: TA100 and TA1537 with and without metabolic activation and TA1535 without metabolic activation.

Under the conditions of this study, the test material ITX was determined to be mutagenic to Salmonella typhimurium TA98 in the absence of metabolic activation.

The potential of the test material ITX to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100). The test was performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

A preliminary dose range finding test was performed at 5, 50, 500 and 5000 µg/ plate. The test material was not toxic towards the tester strains. Therefore 5000 μg/plate was chosen as the top dose level in the mutation tests.

The main test was performed at 50, 150, 500, 1500 and 5000 µg/ plate both with and without metabolic activation (S9 mix). Agar plates were incubated for 72 hours at 37 °C. Colonies were counted using a Biotran Automatic Colony Counter, and the mean number of revertant colonies per treatment group assessed.

The test material was not toxic towards the tester strains. No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with the test material at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Under the conditions of the study, it is concluded that the test material ITX is not mutagenic in the Salmonella typhimurium reverse mutation assay.

The potential of the test material ITX to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B14 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

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 test material ITX 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).

The genetic toxicity of the test material ITX was investigated in accordance with the standardised guideline OECD 471. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2urvA, were subjected to the test material using the plate incorporation method. Doses of 61.7, 185.2, 555.6, 1666.7, 5000.0 µg/plate were tested with or without metabolic activation, in the form of S9-mix. All plates were incubated at 37 °C for 48hours. After the incubation period, the number of revertant colonies in each plate was counted.

The precipitate appeared at 1666.7 and 5000.0 μg per plate for five tester strains with or without S9-mix, but it didn't interfere the colony counting. For TA98, the number of revertant colonies induced by the test material exceeded that of negative control more than two times at 1666.7 and 5000.0 μg per plate without S9-mix, and it demonstrated obvious concentration dependent relationship.

Under the conditions of this study the genotoxic effect of the test material ITX was positive to Salmonella typhimurium TA 98 without metabolic activation. The test material ITX was negative to TA 98 with metabolic activation and to all other tested strains with or without metabolic activation.

The potential of the test material ITX to cause chromosome aberrations in cultured mammalian cells was investigated in accordance with the standardised guideline OECD 473. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

CHL/IU cells were treated with the test material according to the following:

- Test 1 (with metabolic activation) for exposure time 6 hours and harvest time 24 hours at 37.5, 70 and 150 µg/mL;

- Test 1 (without metabolic activation) for exposure time 6 hours and harvest time 24 hours at 15, 30 and 60 µg/mL;

- Test 2 (without metabolic activation) for exposure time of 24 hours and harvest time 24 hours; and

- Test 3 (without metabolic activation) for exposure time 48 hours and harvest time 48 hours.

Metabolic activation was provided in the form of Induced rat liver microsomal fraction. The test material was prepared in DMSO and this was used as the solvent control. Mitomycin C was used as a positive control in the absence of exogenous metabolic activation and Cyclophosphamide was used for the presence of exogenous metabolic activation. Cytotoxicity was determined with or without metabolic activation using viable cell counts.

In the cytotoxicity measurement, it was determined that concentrations of the test material at 60 µg/mL, 40 µg/mL and 10 µg/mL respectively for 6 hours, 24 hours and 48 hours without metabolic activation and at 150 µg/mL with metabolic activator could result in a significant inhibition of cell growth by 50 percent.

Without metabolic activation, the test material did not cause structural chromosome aberration in CHL cells exposed for 6 hours, 24 hours and 48 hours respectively, but it could induce the increase of gaps in CHL cells treated for 48 hours. The test material didn't induce structural chromosome aberration in CHL cells with metabolic activator.

The test material ITX caused a significant increase of polyploidy in CHL cells at 60 µg/mL in the case of CHL cells exposed for 6 hours without metabolic activation (P<0.05). No other abnormality was observed.

In this study, it was observed that without metabolic activation, polyploidy of CHL cells increased significantly when exposed for 6 hours at 60 µg/ml of test material, while this case didn't occur when exposed for 24 hours and 48 hours, respectively. Since the polyploidy ratio is as low as 2.5 % and there was no obvious dose-response relationship it is therefore concluded that the 6 hours results may not be related to action of the test material.

Under the conditions of this study, the test material ITX is not clastogenic in either the presence or absence of metabolic activation.

The genetic toxicity of the test material ITX was investigated in the mammalian gene mutation test in accordance with the standardised guideline OECD 476. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

L5178Y mouse lymphoblastoid cells were treated with the test material for 48 hours at the following concentrations: 20.0, 30.0, 45.0 and 67.5 µg/mL. Cells were treated both in the presence and absence of metabolic activation in the form of Induced rat liver microsomal fraction (S9). Trifluorothymine deoxyribose (3 µg/mL) was used as the selective agent.

The conclusion of the test was valid since the results of PEO, PE2, MF and SC of the negative and positive control were in normal range.

It was observed the mutant frequency increased at 20.0, 30.0 and 45.0 µg/mL. The outcome can't be observed in 67.5 µg/mL group without metabolic activation because 67.5 µg/mL was too high so the cells at that concentration were dead. There was a concentration-related increase in mutant frequency from 20.0 to 45.0 µg/mL without and with metabolic activation, and the scores of small clone ascended with the concentrations in the treatment groups.

Under the conditions of this study the test material ITX was observed to be positive in the Mammalian Gene Mutation Test.

Genetic toxicity in vivo

The genetic toxicity in vivo of the test material ITX was investigated in a study performed in accordance with the standardised guidelines OECD 474 and EU Method B.12 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was investigated in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The test material was formulated in propylene glycol.

Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 2000 (two groups), 1000 (one group), and 500 (one group) mg/kg body weight. The animals of all dose levels showed no abnormalities after dosing.

Bone marrow of the groups treated with the test material was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control group was harvested 24 and 48 hours after dosing, respectively. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test material.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of the test material on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

Under the conditions of this study it is concluded that the test material ITX is not mutagenic in the micronucleus test.

The DNA repair inducing ability of the test material ITX in male Wistar rat hepatocytes, measured as unscheduled DNA synthesis (UDS) was investigated in accordance with the standardised guidelines OECD 486 and EU Method B39, under GLP conditions.

The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was prepared in propylene glycol. Dose groups received a single oral dose of the test material. Hepatocytes were sampled 2-4 or 12-16 hours after dosing with 2000 and 1000 mg/kg body weight (b.w.). All animals showed no abnormalities after dosing, except for one vehicle control animal, which was lethargic and had a hunched posture just prior to the perfusion.

Corresponding vehicle treated groups served as negative controls (vehicle was propylene glycol). Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg b.w.) or 2-acetylaminofluorene (2-AAF, 50 mg/kg b.w.) were harvested 2-4 or 12-16 hours after dosing respectively. Two slides per animal and three animals per data point were examined.

As a result of oral dosing with the test material the net nuclear grain count (NNG) per coverslip and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG≥5), both per individual animal and for the group average, revealed no significant increase at any dose.

The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system ITX was functioning correctly.

Under the conditions of this study the test material was not genotoxic to male Wistar rats.

Justification for classification or non-classification

Based on the available in vitro and in vivo data of the substance 2-Isopropylthioxanthone (CAS 5495-84-1, EC 226-827-9), the structurally related registration substance 2,4-diethyl-9H-thioxanthen-9-one (CAS 82799-44-8, EC 280-041-0) is considered to be not genotoxic and does not require classification according to Regulation (EC) No. 1272/2008.