Diphenyl[4-(phenylsulfanyl)phenyl] sulfonium hexafluoroantimonate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Positive (Mutagenic); OECD 471;P.W. Thompson. (2003)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August - 07 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2000

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary: (with and without S9) - 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Toxicity was observed from 500µg/plate and above TA100 & WP2uvrA strains.

Main study I (Plate incorporation method):
TA100 and TA1535 (presence of S9-mix) - 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA98, TA1535, TA1537 & E. coli (absence of S9-mix) - 5, 15, 50, 150, 500 and 1500 µg/plate

Main study II (Plate incorporation method):
TA100 and TA1535 (presence of S9-mix) - 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate
TA98, TA1535, TA1537 & E. coli (absence of S9-mix) - 5, 15, 50, 150, 500 and 1500 µg/plate

Main study III (Plate incorporation method):
TA1537 ( with /without S9) - 50, 100, 150 and 200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation
- Cell density at seeding (if applicable): n/a

DURATION
- Preincubation period: n/a
- Exposure duration: 48 h (both methods) at 37°C
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity: Measurement of toxicity and precipitation

OTHER EXAMINATIONS: n/a
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER:

Rationale for test conditions:

The study was based on the in vitro (pre-incubation and plate incorporation methods) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.very times): not stated

Evaluation criteria:

The test material is considered positive if it induced a reproducible dose-related and statistically (Dunnett’s Method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett’s Method of linear regression (5)

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (preincoporation method) the test item caused a toxicity in TA100 and WP2uvrA at 500 μg/plate and over in the absence and presence of S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Remarks on result:

other: Not mutagenic

Table 1. Summary results: Range-finder study with and without metabolic activation

Test period	12 - 20^THSep 2002
With or without S9 mix	Dose mg/plate	Mean No. Revertant colonies/plste
		Base-pair substitution type			Frame-shift type
		TA100+	TA1535	WP2uvrA	TA98	TA1537
-S9	0	74	15	20	25	9
	1.5	81	NT	NT	NT	NT
	5	83	15	21	16	9
	15	67	16	16	15	8
	50	85	16	19	21	17*
	150	53	10	19	18	180$$$
	500	0T	0T	15T	12T	0T
	1500	0T	0T	0T	0T	0T
+S9	0	82	14	28	27	17
	1.5	79	11	NT	NT	NT
	5	78	14	19	33	12
	15	85	14	28	27	9
	50	87	13	16	25	17
	150	57T	13	22	35	90$$$
	500	0T	0T	14T	17T	0T
	1500	0T	0T	0T	0T	0T
Positive control -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Dose mg/plate	3.0	5.0	2.0	0.2	80.0
	No. Colonies/plate	449	223	362	218	1377
Positive control +S9	Name	2AA	2AA	BP	2AA	2AA
	Dose mg/plate	1.0	2.0	10.0	5.0	2.0
	No. Colonies/plate	1416	433	907	325	603

 

 

Table 2. Summary results: Main study with and without metabolic activation

Test period	26 -29^thSept 2002
With or without S9 mix	Dosemg/plate	No. Revertant colonies/plste
		Base-pair substitution type			Frame-shift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-S9	0	100	24	27	21	12
	1.5	94	NT	NT	NT	NT
	5	83	11	23	18	9
	15	83	14	23	22	5
	50	69	14	19	17	11
	150	57	10	13	20	53$$
	500	0T	0T	15T	11T	0T
	1500	0T	0T	0T	0T	0T
+S9	0	89	14	30	35	14
	1.5	77	15	NT	NT	NT
	5	72	12	25	28	11
	15	85	10	26	25	13
	50	71	12	24	31	9
	150	49	17	22	30	45$$
	500	0T	0T	16T	14T	0T
	1500	0T	0T	0T	0T	0T
Positive control -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Dosemg/plate	3.0	5.0	2.0	0.2	80.0
	No. Colonies/plate	386	338	423	309	3003
Positive control +S9	Name	2AA	2AA	BP	2AA	2AA
	Dosemg/plate	1.0	2.0	10.0	5.0	2.0
	No. Colonies/plate	1195	450	621	268	550

 

  Table 3. Summary results: Confirmatory study with and without metabolic activation (Plate incorporation method)

Test Period		4 – 7 Oct 2002	Test Period		4 – 7 Oct 2002
With or without S9	Test Item Dose mg/plate	With or without S9	With or without S9	Test Item Dose mg/plate	Mean no. colonies per plate
		Frameshift type TA1537			Frameshift type TA1537
-S9	0	5	+S9	0	20
	50	6		50	15
	100	19$$$		100	18
	150	12**		150	9
	200	6		200	9
Positive control -S9	Name	9AA	Positive control +S9	Name	2AA
	Dosemg/plate	80		Dosemg/plate	2
	colonies per plate	2332		colonies per plate	140

9AA = 9-Aminoacridine

2AA = 2-Aminoanthracene

BP  = Benzo (a)pyrene

C= Contaminated

NT = Not tested at this dose level

T = Partial absence of background lawn

** p =< 0.01

$$$ =< 0.005

Conclusions:

Under the conditions of the test the substance was considered to be weakly mutagenic in TA1537 with and without metabolic activation.

Executive summary:

OECD 471 (2003): The test substance, CPI-2, was tested to evaluate its mutagenic potential using both pre-incubation and plate incorporation methods by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium  (TA98, TA100, TA1535 & TA1537) and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 5 - 1500 μg per plate, based on data generated in preliminary study where growth inhibition was observed at 500 μg per plate in all TA100 and TA1535 strains.  An insufficient number of non-toxic dose levels were achieved in tester strains TA100 (+/- S9) and TA1535 (+S9), therefore, the experiment was repeated with dose range of 1.5 - 1500 μg per plate. A third confirmatory experiment was conducted using TA1537 at concentration of 50, 100, 150 & 200 μg per plate in an effort to confirm both dose-response relation and reproducibility using the direct plate and pre-incubation methods. All criteria for a valid study were met as described in the protocol.

Visible reduction in the growth of bacterial background lawn and or a decrease in revertant colony frequency to TA100 at and above 150 μg per plate and to all of the remaining tester strains from 500 μg per plate. No precipitation was observed at any dose level with and without S9 mix. In the range finder study, significant increase in revertant colony frequency to TA1537 at 150 μg per plate  (+/- S9) and at 50 μg per plate  (-S9). Similar effects was also observed in the main study at 150 μg per plate although the increase was slightly lower than dose range finder.  The Confirmatory result did not show any statistical increase in revertant colony using the pre-incubation but signifigant statistical increase was observed using the plate incorporation at doses of 100 and 150 μg per plate (-S9) and visible reduction in bacterial background growth at 200 μg per plate.

No dose dependent increases were observed in other tester strain  with or without metabolic activation. The test item was considered weakly mutagenic in bacterial reverse mutation assay  without metabolic activation in tester strain TA1537.

Under the conditions of this study, test item met the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixture.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No conclusion can be made on the mode of action of the substance and its relevance to human health. Further information is required to conclude on genotoxicity endpoint as the in vitro bacterial mutation only focus one mode of action and further mode of actions will need to evaluated in order to determine the mutagenicity potential of the substance.

Additional information

OECD 471 (2003): The test substance, CPI-2, was tested to evaluate its mutagenic potential using both pre-incubation and plate incorporation methods by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium  (TA98, TA100, TA1535 & TA1537) and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay were 5 - 1500 μg per plate, based on data generated in preliminary study where growth inhibition was observed at 500 μg per plate in all TA100 and TA1535 strains.  An insufficient number of non-toxic dose levels were achieved in tester strains TA100 (+/- S9) and TA1535 (+S9), therefore, the experiment was repeated with dose range of 1.5 - 1500 μg per plate. A third confirmatory experiment was conducted using TA1537 at concentration of 50, 100, 150 & 200 μg per plate in an effort to confirm both dose-response relation and reproducibility using the direct plate and pre-incubation methods. All criteria for a valid study were met as described in the protocol.

Visible reduction in the growth of bacterial background lawn and or a decrease in revertant colony frequency to TA100 at and above 150 μg per plate and to all of the remaining tester strains from 500 μg per plate. No precipitation was observed at any dose level with and without S9 mix. In the range finder study, significant increase in revertant colony frequency to TA1537 at 150 μg per plate  (+/- S9) and at 50 μg per plate  (-S9). Similar effects was also observed in the main study at 150 μg per plate although the increase was slightly lower than dose range finder.  The Confirmatory result did not show any statistical increase in revertant colony using the pre-incubation but signifigant statistical increase was observed using the plate incorporation at doses of 100 and 150 μg per plate (-S9) and visible reduction in bacterial background growth at 200 μg per plate.

No dose dependent increases were observed in other tester strain  with or without metabolic activation. The test item was considered weakly mutagenic in bacterial reverse mutation assay  without metabolic activation in tester strain TA1537.

Under the conditions of this study, test item met the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixture.

Justification for classification or non-classification

In conclusion based on available data, further information is required to conclude on genotoxicity endpoint as the in vitro bacterial mutation only focus one mode of action and further mode of actions will need to evaluated in order to determine the mutagenicity of the test item. Therefore, the classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixture is considered inconclusive.