lithium 3-((3,4-dicyanophenyl)sulfonyl)propane-1-sulfonate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

S193308 was negative in an in vitro Ames test but postive in an in vitro mammalian chromosome aberration test (IVC). S193308 is not structurally alerting for mutagenicity so the positive response in the IVC was unexpected. An in vivo mouse micronucleus assay gave a negative result and this in association with the Ames result is considered to mitigate the IVC result and consequently S193308 is not considered to be mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro cytogenicity study in mammalian cells

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 21 May 2012 and 15 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 10 July 2012. Date of Signature: 07 September 2012

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Cell Growth Inhibition Test:
4(20)-hour without S9: 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 µg/ml.
4(20)-hour with S9: 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 µg/ml.
20-hour without S9: 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 µg/ml.

Experiment 1
4(20)-hour without S9: 0*, 100, 200, 400, 800*, 1600*, 3200*, µg/ml.
4(20)-hour with S9: 0*, 100, 200, 400, 800*, 1600*, 3200*, µg/ml.

Experiment 2
24-hour without S9: 0*, 1250, 100, 200*, 400, 800*, 1600*, 3200 µg/ml.
4(20)-hour with S9: 0*, 100, 200, 400, 800*, 1600*, 3200* µg/ml.

* Dose levels selected for metaphase analysis

Vehicle / solvent:

Vehicle: Eagle's minimal essential medium with HEPES buffer (MEM)

Justification for choice of solvent/vehicle:
The test item was soluble in Eagle's minimal essential medium with HEPES buffer (MEM) at 32 mg/ml in an in-house solubility check. .

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 Migrated to IUCLID6: (CP)EXAMPLE:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

:

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 Migrated to IUCLID6: (MMC)

Details on test system and experimental conditions:

METHODS OF APPLICATION:
In medium

DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours

SELECTION AGENT (MUTATION ASSAYS):
No selection agent selected

SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS:
Duplicate cultures

NUMBER OF CELLS EVALUATED:
100/culture

DETERMINATION OF CYTOTOXICITY
-Method:
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells

OTHER:
none

Evaluation criteria:

A positive response is recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent
control, either with or without a clear dose-response relationship. For modest increases in aberration frequency, a dose response relationship is generally
required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle
control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

24-hour exposure without S9 only

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to information on results and attached tables.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 12.5 to 3200 µg/ml. The maximum dose was based on the maximum recommended dose level, 10 mM concentration. There was no precipitate observed in the parallel blood-free cultures at the end of the exposure period. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3200 µg/ml in all exposure groups. However, in the 24-hour continuous cultures qualitative assessment showed that there was a marked dose-related reduction in the numbers of metaphase cells observed at and above 1600 µg/ml. The mitotic index data are presented in Table 1 (see attached background material section), and it can clearly be seen that there was dose-related marked toxicity observed in the 24-hour exposure group.

Dose selection was based on the maximum recommended dose level (3200 µg/ml) in all exposure groups in both experiments.

Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of Isostearamide DEA (µg/ml)
4(20)-hour without S9 0*, 100, 200, 400, 800*, 1600*, 3200*, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 100, 200, 400, 800*, 1600*, 3200*, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide

The qualitative assessment of the slides determined that there was modest toxicity present and that there were metaphases suitable for scoring present at the maximum test item dose level tested, 3200 µg/ml, in both exposure groups. No precipitate of the test item was observed at the end of exposure, in either exposure group.

The mitotic index data are given in Table 2 (see attached background material section). These data show there was a modest dose-related reduction in Mitotic Index of the 4(20)-hour exposure in the absence of S9, with a maximum value of 71% at 3200 µg/ml. However, near optimum toxicity was observed in the 4(20)-hour exposure in the presence of S9 (Mitotic Index 54%) where dose-related reductions were also observed.

The maximum dose level selected for metaphase analysis was, therefore, 3200 µg/ml in both the presence and absence of S9. The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Tables 4 and 5. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test – Experiment 2
The dose levels of the controls and the test item are given in the table below:

Group Final concentration of Isostearamide DEA (µg/ml)
24-hour without S9 0*, 50, 100, 200*, 400, 800*, 1600*, 3200, MMC 0.2*
4(20)-hour with S9 (1%) 0*, 100, 200, 400, 800*, 1600*, 3200*, CP 5*

*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 1600 µg/ml in the absence of S9 and at 3200 µg/ml in the presence of S9. No precipitate of the test item was observed at the end of exposure, in either exposure group.

The mitotic index data are given in Table 3 (see atached background material section). They confirm the qualitative observations in that inhibition of mitotic index was observed, and that dose related mitotic inhibition was observed in the absence of S9. The Mitotic Indices of 88%, 58% and 48% were observed at 200, 800 and 1600 µg/ml, therefore, optimum toxicity was achieved at the highest dose level scored. There were no scorable metaphases present at 3200 µg/ml in this exposure group. In the presence of S9, near optimal toxicity was observed at 3200 µg/ml (Mitotic Index 56%).

The maximum dose level selected for metaphase analysis was therefore, 1600µg/ml and 3200 µg/ml in the absence and presence of S9, respectively.The chromosome aberration data are given in Table 6 and Table 7 (see attached background material section). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced statistically significant increases in the frequency of cells with aberrations in the 24-hour continuous exposure group at 800 and 1600 µg/plate (P = 1.8 X10-3 and P<10-8, respectively). Both dose levels were within the optimal maximum level of 50% mitotic inhibition, with 42 and 52% reductions respectively. However, at 800 µg/ml the response was lop-sided in that almost all of the aberrations were observed in the B culture only, which was marginally more toxic, with predominantly break-type aberrations. The distribution of aberrations observed at 1600 µg/ml was more even between both cultures but all of the aberrations seen were break-type aberrations. It should be noted that there was a marked increase in the numbers of gaps as well in both dose levels. With only a couple of exchange type aberrations being recorded at 800 µg/ml, and no response in the lowest dose level, it is possible that the observed aberrations are due to a cytotoxic mechanism rather than a true genotoxic mechanism. Therefore, the response may have little biological relevance. The test item, however, did not induce any statistically significant increases in the frequency of cellswith aberrations in the 4(20)-hour exposure group, in the presence of S9, which included a dose level that was within the optimal 50% mitotic inhibition.

The polyploid cell frequency data are given in Tables 6 and 7 (see atached background material section). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation 24-exposure group only

The test item, S193308, was considered to be clastogenic to human lymphocytes in vitro following 24 hours continuous exposure in the absence of metabolic activation only under the conditions of this test.

Executive summary:

Introduction. This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design was also compatible with the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group	Final concentration of test item (µg/ml)
4(20)-hour without S9	100, 200, 400, 800, 1600, 3200
4(20)-hour with S9 (2%)
24-hour without S9	50, 100, 200, 400, 800, 1600, 3200
4(20)-hour with S9 (1%)	100, 200, 400, 800, 1600, 3200

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced statistically significant dose-related increases in the frequency of cells with aberrations in the 24-hour continuous exposure group only at 800 and 1600 µg/plate. Both dose levels were within the optimal 50% mitotic inhibition (Mitotic Indices 62% and 58%, respectively). However, the test item did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)‑hour exposure groups, in either of two separate experiments using a dose range that generally included a dose level that was within the optimal 50% mitotic inhibition.

Conclusion. The test item, was considered to be clastogenic to human lymphocytesin vitrofollowing 24‑hours continuous exposure in the absence of metabolic activation only under the conditions of this test.

 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 30 May 2012 and 10 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

See Appendix 3

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test; 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Expt 1 (plate incorporation): 50, 150, 500, 1500 and 5000 µg/plate.
Expt 2 (pre-incubation): 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

Vehicle: Dimethyl sulphoxide

Justification for choice of solvent/vehicle: The test item was partially soluble in sterile distilled water at 50 mg/ml but fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

Experiment 1
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Experiment 2
METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2
- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks. All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Combined historical negative and solvent control ranges for 2010 and 2011 are presented in Appendix 2 (see attached background material section). All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml. Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure, the integrity of the S9-mix and should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1: A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2: A reproducible increase at one or more concentrations.
3: Biological relevance against in-house historical control ranges.
4: Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5: Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard Deviation
Dunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. There was also no evidence of excessive contamination. The culture density for each bacterial strain used in each experiment was also checked and considered acceptable. Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4. Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1 (see attached background material section).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

RESULTS

The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9‑mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9‑mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	87	96	94	107	106	103	100	95	89	98	74
+	TA100	96	81	85	82	86	97	74	67	97	81	73
-	WP2uvrA	32	39	25	34	32	36	40	30	40	36	28
+	WP2uvrA	24	26	21	24	34	33	26	25	36	24	27

 

 

Mutation Test

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1

 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
84		20		39		21		13
83	(85)	24	(20)	44	(40)	29	(26)	13	(13)
87		17		36		28		12

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
92		10		30		13		12
88	(90)	10	(15)	29	(26)	19	(16)	11	(10)
90		25		18		15		7

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, attachments

References:

Ames B N, McCann J and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.

Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173 - 215.

Mortelmans K and Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60.

Green M H L and Muriel W J (1976), Mutagen Testing Using TRP+ Reversion in Escherichia coli, Mutation Research, 38, 3- 32.

Maron D M, Katzenellenbogen J and Ames B N (1981), Compatibility of organic solvents with the Salmonella/Microsome Test, Mutation Research, 88, 343-350.

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92.

Mahon G A T et al (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report, 26-65.

Conclusions:

Interpretation of results (migrated information):
negative

The test item, S193308, was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item, S193308, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. 

Results.The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion.The test item,S193308, was considered to be non-mutagenic under the conditions of this test.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

S193308 was negative in an in vitro Ames test but postive in an in vitro mammalian chromosome aberration test (IVC). S193308 is not structurally alerting for mutagenicity so the positive response in the IVC was unexpected. An in vivo mouse micronucleus assay gave a negative result and this in association with the Ames result is considered to mitigate the IVC result and consequently S193308 is not considered to be mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of the study was performed between 13 May 2013 and 03 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997).

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The test animals were albino Hsd: ICR (CD-1®) strain mice obtained from Harlan UK. At the start of the main test the mice weighed 23 to 30g and were approximately six to ten weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

intraperitoneal

Vehicle:

Arachis oil for animals dosed intraperitoneally with test susbtance.

Details on exposure:

Range-Finder Test

A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The criteria for dose level selection is ideally the maximum tolerated dose level, or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity test was also used to determine if the main test was to be performed using both of the sexes or males only.

All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of dosing. Animals were observed 1, 3 and 4 hours after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed. As it was not possible to confirm systemic absorption by the oral route following dosing at 2000 mg/kg, further dosing was undertaken via the intraperitoneal route.

Micronucleus Test

Groups, each of seven male mice, were dosed once only via the intraperitoneal route with the test item at 800, 400 or 200 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 800 mg/kg was killed after 48 hours. In addition, two further groups of male mice were included in the study; one group (seven mice) was dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a second group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test. The vehicle and positive control group animals were killed 24 hours following dosing.

Frequency of treatment:

Once only

No. of animals per sex per dose:

Seven male mice were used per dose group and vehicle control. The postive control group consisted of five males.

Control animals:

yes, concurrent vehicle

other: Positve control

Positive control(s):

Cyclophosphamide dosed orally was used as the positive control.

Tissues and cell types examined:

Bone Marrow.

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a square root of (x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

one death and clinical signs observed in animals dosed via the intraperitoneal route at 1000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Range- Finder Toxicity Test

In animals dosed with the test item at 2000 mg/kg via the oral route, no clinical signs were observed throughout the 48 hour exposure period. The route of administration was therefore changed to the intraperitoneal route to maximise exposure to the test item.

In animals dosed with the test item at 1000 mg/kg via the intraperitoneal route, a premature death occurred and the following clinical signs were observed: hunched posture, lethargy, ataxia, splayed gait, elevated tail, clonic convulsions, and tonic convulsions. These indicated that systemic absorption had been achieved. In animals dosed with the test item at 300 mg/kg, clinical signs were not observed. In animals dosed with the test item at 600 and 800 mg/kg, the clinical signs hunched posture and ptosis was observed. Therefore, with evidence of excessive toxicity at 1000 mg/kg, the maximum dose level in the main test was set at 800 mg/kg, the considered maximum tolerated dose level, with 400 and 200 mg/kg as the two lower dose levels using the intraperitoneal route of administration.

The test item showed no marked difference in its toxicity to male or female mice; therefore the main test was performed using male mice only.

Micronucleus Test

There were no premature deaths seen in any of the dose groups. The clinical signs hunched posture and ptosis were observed in animals dosed with the test item at 800 mg/kg in both the 24 and 48-hour dose groups.

A statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 200 mg/kg dose group and, although not statistically significant, a modest decrease was also observed in the 24-hour 400 mg/kg dose group. However, the reductions were part of an inverse dose related response and, therefore, may be considered to be artefactual and of no toxicological significance.

The observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

See attached background material section for tables of results.

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy. The method was designed to meet the requirements of the following guideline:

OCED Guideline for Testing of Chemicals No 474 " Mammalian Erthrocyte Micronucleus Test (adopted 21st July 1997)

Method

Groups of seven male mice, were dosed once only via the intraperitoneal route with the test item at 800, 400 or 200 mg/kg. One group of mice from each dose level was killed 24 hours following treatment and a second group dosed with test item at 800 mg/kg was killed after 48 hours. A vehicle control (arachis oil) group and a postive control (cyclophosphamide) group were also included in the study.

Results

A statistically significant decrease in the PCE/NCE ratio was observed in the 24 -hour 200 mg/kg test item group and although not statistically significant, a modest increase was also observed in the 24 -hour 400 mg/kg dose group. However, the reductions were part of an inverse dose related response and considered to be artifactual and of no toxicological significance. The observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positve control group responded as expected.

Conclusion

The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

S193308 was negative in an in vitro Ames test but postive in an in vitro mammalian chromosome aberration test (IVC). S193308 is not structurally alerting for mutagenicity so the positive response in the IVC was unexpected. An in vivo mouse micronucleus assay gave a negative result and this in association with the Ames result is considered to mitigate the IVC result and consequently S193308 is not considered to be mutagenic.