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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

700-777-2

Cas Number:

667889-11-4

Molecular formula:

C11H9N2O5S2Li

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: S193308
Description: White powder
Purity: 98.9%
Batch number: 22 (Bag 3)
Date received: 02 May 2012
Expiry date: Not supplied
Storage conditions: Room temperature in the dark

Method

Target gene:

Not applicable.

Species / strain

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. .

Controls

Controls 1

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:

Controls 2

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.

Results and discussion

Test results

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'.

Applicant's summary and conclusion

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.