[(2-chlorophenyl)methylene]malononitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

As presented by NTP (1990) : CS2 was tested for induction of gene mutations in a total of five strains of Salmonella typhimurium in two different laboratories using a preincubation protocol with and without Aroclor 1254- induced male Sprague Dawley rat or Syrian hamster liver S9 (Zeiger et al., 1987). In one laboratory, an equivocal response was noted in strain TA97, but only in the presence of 30% hamster liver S9; in the other four strains tested (TA98, TA100, TA1535, and TA1537), no mutagenic response was observed with or without S9 (10% or 30%). In the other laboratory, an equivocal response occurred with strain TA100 in the absence of S9 only; CS2 was clearly negative for gene mutation induction in all other strains tested in this laboratory (TA98, TA1535, and TA1537) with or without S9. CS2 induced trifluorothymidine resistance in mouse L5178YPTK lymphoma cells at the highest non- lethal dose tested (2.5 pg/ml) in each of two trials conducted in the absence of S9; it was not tested with S9 (McGregor et al., 1988). In cytogenetic tests with Chinese hamster ovary cells, CS2 induced both sister chromatid exchanges (SCEs) and chromosomal aberrations with and without Aroclor 1254-induced male Sprague Dawley rat liver S9. For both the SCE and the aberration tests, a delayed harvest protocol was used to offset CS2-induced cell cycle delay at each of the dose levels at which a positive response was demonstrated.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Salmonella/microsome assay for mutagenicity was performed with salmonella Typhimurium TA 100, according to Ames et al. (1975).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not specified

Species / strain / cell type:

S. typhimurium TA 100

Test concentrations with justification for top dose:

Test without metabolic activation : 5 concentrations : between 0 to 2000 µg/plate
Test with metabolic activation : several concentrations between 0 to 3500 µg/plate

Vehicle / solvent:

Not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

sodium azide : 1.0 µg or 2.5 µg/plate - benzoapyrène : 5.0 µg/plate

Positive control substance:

sodium azide

benzo(a)pyrene

Details on test system and experimental conditions:

The assays were performed without pre-incubation.

Rationale for test conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

With rat liver homogenate the bacteriotoxicity of CS was slightly stronger than without it.

Conclusions:

No mutagenic effect was detected using CS either with or without rat liver homogenate (S-9 mix)

Executive summary:

In a study which methodology is comparable to OECD 471 (Ames test), no mutagenic effect was detected using CS either with or without rat liver homogenate (S-9 mix)

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Maron DM, Ames BN (1983): Revised methods for the Salmonella mutagenicity test. Mutat Res 113: 173-21s.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Suppliers : Aberdeen Proving Gd.
Purity : 99.9%
Testing Lab : MIC, SRI

Target gene:

Histidin

Species / strain / cell type:

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

Remarks:

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat livers. The S-9 mixes were prepared immediately prior to use and contained either 10%

Test concentrations with justification for top dose:

Five doses of the chemical were tested in triplicate :
- without metabolic activation (1 ; 3 ; 10 ; 33 ; 100 µg/plate)
- with metabolic activation (3 ; 10 ; 33 ; 100 ; 333 µg/plate)

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-phenylenediamine (TA98 without metabolic activation) ; 2-aminoanthracene (all strains, with metabolic activation)

Details on test system and experimental conditions:

Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed,

Evaluation criteria:

An individual trial was judged :
- mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen.
- questionable (?) if a doserelated increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.
The distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response are highly subjective.
A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.
A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.
The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity

Statistics:

Not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not specified

Conclusions:

Results were negative for the strains : TA98, 1535 and 1537. Results were qualified of "equivocal to weakly positive responses" by NTP in report Tr 377 (1990) for the strain TA100 with, or without metabolic activation.

Executive summary:

In a study in vitro which methodology is comparable to OECD 471 (Ames test), results were negative for the strains : TA98, 1535 and 1537. Results were qualified of "equivocal to weakly positive responses" by NTP in report Tr 377 (1990) for the strain TA100 with, or without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Principles of method if other than guideline:

Responses of the L5178Y tk+/tk’ Mouse Lymphoma Cell Forward Mutation Assay, procedures based upon those described by Clive and Spector [Mutat Res 44:269-278, 19751] and Clive et al [Mutat Res 59:61-108, 1979]

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Test item supplied from the National Toxicology Program Chemical Repository, Radian Corporation (Austin, TX).purity level not specified in the publication.

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y tk +/- mouse lymphoma cells, clone 3.7.2C
Obtained from : Dr. D. Clive, Burroughs Wellcome Co. (Research Triangle Park, NC)
Storage : in liquid nitrogen
Sterility : no mycoplasma
Fischer’s medium (designated Fo) was supplemented with 2 mM L-glutamine, sodium pyruvate (110 pg/ml), 0.05% pluronic F68, antibiotics, and 10% heat-inactivated donor horse serum (v/v) (designated Flop). On a single occasion, within 1 week of the start of an experiment, cultures were purged of tk+/ tk- mutants by exposure for 1 day to F10P containing THMG (thymidine, 6 pg/ml; hypoxanthine, 5 pg/ml; glycine, 7.5 pg/ml; and methotrexate, 0.1 pg/ml), then for 3 days to Flop containing THG only, (ie, THMG without methotrexate).

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

5-trifluorothymidine (TFT), 3 pg/ml.

Metabolic activation:

with and without

Metabolic activation system:

Postmitochondrial supernatant fractions of liver homogenates (S9) were prepared from 200 g,male F344 rats, obtained from the Frederick Cancer. Research Center (Frederick, MD). Preparation S9 : rats injected intraperitoneally with Aroclor 1254 (500 mg/kg)

Test concentrations with justification for top dose:

5 concentrations (0 ; 0.3125 ; 0.625 ; 1.25 ; 2.5 µg/mL)
2 cultures per concentration

Vehicle / solvent:

Dimethyl sulphoxide (DMSO), which was analytical grade obtained from BDH Limited (Poole, Dorset, England). 10 µL/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

Experimental design :

The first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/ml unless a much lower concentration was indicated by the poor solubility of a compound. This test was followed by at least two experiments in the absence of S9 mix.
If no clear positive response was observed, then two experiments were performed in the presence of S9 mix. Test compound concentrations were primarily two-fold dilutions from the highest testable concentration, as estimated from the toxicity test. The decision not automatically to test in the presence of S9 mix if a compound was mutagenic without it was based solely on economic grounds. Subsequent, more detailed study of a compound and its mechanism of action would lead to a different program design.

Mutation experiment :

Exposure. Each exposed culture consisted of 6 x 10.6 cells in a final volume of 10 ml F5p in a 30-ml screw-cap plastic tube (Sterilin Ltd., Hounslow, Middlesex, England). This tube was incubated for 4 hr on a horizontal axis roller drum rotating at 10 rpm. At the end of the incubation time the cells were sedimented by centrifugation at 500g for 10 min, washed, and finally resuspended in 20 ml F10P. These cell suspensions (3 x 10.5 cells/ml) were incubated for a 2-day expression period, the cell population density being adjusted back to 20 ml of 3 X 10.5 cells/ml after 24 hr. After 48 hr, the cell population densities were estimated and culture volumes containing 3X 10.6 cells adjusted to 15 ml with F10P, giving a cell population density of 2 X 10.5 cells/ml.

Cloning efficiency. A 0.1-ml sample of the cell suspension was withdrawn and diluted 1:lOO. Three 0.1-ml samples (200 cells) of the diluted cultures were transferred to 30-ml tubes, mixed with 25 ml of cloning medium (Fischer’s medium containing 20% heat-inactivated horse serum, ie, F2%) containing 0.35% Noble agar at 37°C and poured into 90-mm Petri plates.

Mutant selection. Three aliquots (each containing lo6 cells) of the remaining culture were distributed to 30-ml tubes, mixed with 20 ml of cloning medium to give final concentrations of 0.35% Noble agar and 3 pg trifluorothymidine/ml at 37”C, then poured into 90-mm Petri plates.

Incubation. The agar was solidified at 4°C for 5-10 min, then the plates were incubated for 11-14 days in 5% CO2:95% air at 37°C.

Colony counting. Colonies were counted using a New Brunswick Biotran I11 Automated Colony Counter, with the colony size discriminator control in the “off’ position

Calculations. Toxicity was expressed as either a reduction of cell population growth in suspension during the expression period or a reduction in cloning efficiency. A measure of the overall toxicity was the relative total growth (RTG).

Rationale for test conditions:

Not specified

Evaluation criteria:

- Positive response (+)
The dose-related trend and the response at one of the three highest acceptable doses were statistically significant.
- Negative response (-)
Two categories were used. In both there was:
(a) No dose-related trend.
(b) No statistically significant response at any dose.
(c) An acceptable positive control response.
- Nontoxic, negative response (=)
There was an RTG among the acceptable doses of >30% (approximately), higher toxicities being unattainable owing to intrinsic properties of either the compound or the system.
- Toxic, negative response (-)
There was either an RTG of <30% (approximately) at the maximum acceptable dose, or the lethal concentration was no greater than 1.5 times a lower concentration at which the RTG was > 30%.
- Inconclusive (i)
There was:
(a) No dose-related trend and a statistically significant dose was any other than one of the highest three doses.
(b) A response which would have been negative, but the lowest RTG at acceptable doses was >35ù
(c) A response which would have been negative, but there were no acceptable positive controls.
- Questionable (?)
There was either:
(a) No dose-related trend, but a statistically significant response occurred at one of the highest
(b) A statistically significant dose-related trend, but none of the acceptable doses was statistically three doses, or significant on its own.

Statistics:

The statistical analysis was based upon the mathematical model proposed for this system [Lee and Caspary, 19831 and consisted of a dose-trend test [Barlow et al, 1972, p 2151 and a variance analysis of
pair-wise comparisons of each dose against the vehicle control. Significant differences from concurrent vehicle control values at the 5% level after variance analysis are indicated by underlining the average mutant fractions at the appropriate concentrations.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not specified

Small, but significant increases in mutant numbers and fractions occurred in two experiments in the absence of S9 mix at a concentration of 2.5 ,ug/ml; the corresponding relative total growth (RTGs) were 18% and 32%.

Conclusions:

The result of the gene mutation test on the substance item was positive without S9 metabolic activation, with a concentration of 2.5 µg of substance per mL.

Executive summary:

The result of the gene mutation test on the substance item was positive without S9 metabolic activation, with a concentration of 2.5 µg of substance per mL.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

CS2 is 94% o-chlorobenzalmalononitrile (CAS No. 2698-41-1) formulated in a mixture of 5% Cab-0-Sil@ colloidal silica and 1% hexamethyldisilizane (CAS No. 999-97-3). No more details are specified.

Target gene:

Not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9

Test concentrations with justification for top dose:

First test serie :
- with S9 : 1 ; 3.3 ; 10 µg/mL
- without S9 : 6 ; 7 ; 8 µg/mL

Second test serie (redone at higher concentrations because of the weakly positive results of the first test with metabolic activation) :
- with S9 : 10 ; 12.5 ; 15 µg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

A delayed harvest protocol was used to offset CS2-induced cell cycle delay at each of the dose levels at which a positive response was demonstrated.

Chinese hamster ovary cells were incubated with study compound or solvent (acetone) and cultured for sufficient time to reach second metaphase division. Cells were then collected by mitotic shake-off, fixed, air dried, and stained.
Percentage change in the value ofSCEskhromosome for exposed culture compared with that for solvent control culture. An increase of20% or more was considered to be a significant response.
- In the absence of S9, Chinese hamster ovary cells were incubated with study compound or solvent for 2 hours'at 37° C. Then BrdU was added, and incubation was continued for 24 hours. Cells were washed, fresh medium containing BrdU and colcemid was added, and incubation was continued for 2-3 hours.
Because some chemicals induce a delay in the cell division cycle, harvest times are occasionally extended to maximize the proportion of second division cells available for analysis (32 or 33 hours instead of 25 hours)
- In the presence of S9, cells were incubated with study compound or solvent for 2 hours at 37° C. Cells were then washed, and medium containing BrdU was added. Cells were incubated for a further 26 hours, with colcemid present for the final 2-3 hours.

Rationale for test conditions:

Not specified

Evaluation criteria:

Percentage change in the value of SCEs chromosome for exposed culture compared with that for solvent control culture. An increase of 20% or more was considered to be a significant response.

Statistics:

Not specified

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

P value for the different tests : P < 0.001

Conclusions:

In CHO cells, CS induced sister chromatid exchanges (SCEs) with and without Aroclor 1254-induced male Sprague Dawley rat liver S9.

Executive summary:

As reported by NTP report Tr377 (1990), in cytogenetic tests (protocol similar to OECD 479) with CHO cells, CS induced sister chromatid exchanges (SCEs) with and without Aroclor 1254-induced male Sprague Dawley rat liver S9.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

CS2 is 94% o-chlorobenzalmalononitrile (CAS No. 2698-41-1) formulated in a mixture of 5% Cab-0-Sil@ colloidal silica and 1% hexamethyldisilizane (CAS No. 999-97-3). No more details are specified.

Target gene:

Not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9

Test concentrations with justification for top dose:

For the test with S9 : 6 ; 7 ; 9 ; 10 µg/mL
For the test without S9 : 20 ; 22,5 ; 25 µg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Compared to the traditional aberration chromosome test, a delayed harvest protocol was used to offset CS-induced cell cycle delay at each of the dose levels at which a positive response was demonstrated.

Cells were arrested in first metaphase by addition of colcemid and harvested by mitotic shake-off, fixed, and stained in 6%Giemsa.

In the absence of S9, cells were incubated with study compound or solvent for 8-10 hours at 37° C. Cells were then washed, and fresh medium containing colcemid was added for an additional 2-3 hours followed by harvest.
In the presence of S9,cells were incubated with study compound or solvent for 2 hours at 37° C. Cells were then washed, me- dium was added, and incubation was continued for 8-10hours. Colcemid was added for the last 2-3 hours of incubation before harvest.

Because of significant chemical-induced cell cycle delay, incubation time prior to addition of colcemid was lengthened to pro-vide sufficient metaphases at harvest. (19h)


Number of cells evaluated :
- 100 cells in the most of the case
- 25 cells in the test with S9 and a concentration of test item = 9 µg/mL
- 10 cells test without S9 and a concentration of test item = 20 and 22.5 µg/mL

- 50 cells for the positive control for the test without metabolic activation (Mitomycin C)
- 20 for the positive control for the test with metabolic activation (Cyclophosphamide)

Rationale for test conditions:

Not specified

Evaluation criteria:

Not specified

Statistics:

Not specified

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Not specified

Conclusions:

In cytogenetic tests with CHO cells, CS induced chromosomal aberration with and without Aroclor 1254-induced male Sprague Dawley rat liver S9

Executive summary:

As reported by NTP report Tr377 (1990), in cytogenetic tests (protocol similar to OECD 473) with CHO cells, CS induced chromosomal aberrations with and without Aroclor 1254-induced male Sprague Dawley rat liver S9.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In accordance with NTP (1990) conclusions, Wild et al. (1983) reported no increase in micronucleated polychromatic erythrocytes in the bone marrow of mice administered CS either by intraperitoneal injection or orally.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Procedures described previously (King et al. 1981)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

purity level not specified

Species:

mouse

Strain:

NMRI

Sex:

not specified

Route of administration:

other: intraperitoneal and per os

Vehicle:

olive oil = negative control

Duration of treatment / exposure:

One administration

Frequency of treatment:

once

Post exposure period:

Expression time 30 to 48 hours

Dose / conc.:

37.8 mg/kg bw (total dose)

Remarks:

intraperitoneal dose - 3/4 of the reported intraperitoneal LD50 in mice (NIOSH 1980)

Dose / conc.:

18.9 mg/kg bw (total dose)

Remarks:

intraperitoneal dose - half of highest dose

Dose / conc.:

226 mg/kg bw (total dose)

Remarks:

per os dose, equivalent to oral LD50 reported by NIOSH 1980

Dose / conc.:

113 mg/kg bw (total dose)

Remarks:

half of highest dose per os

No. of animals per sex per dose:

5 per dose, except for highest dose per os (3 mice survived among 13 as LD50)

Control animals:

yes, concurrent vehicle

Positive control(s):

procarbazine per os and ip

Tissues and cell types examined:

bown marrow erythrocytes

Details of tissue and slide preparation:

point mutation, germinal gene mutations, chromosomal breaks and mitotic chromosome misdistribution

Evaluation criteria:

presence/absence

Statistics:

not specified

Key result

Sex:

not specified

Genotoxicity:

negative

Toxicity:

yes

Remarks:

expected as highest dose per os equivalent to LD50

Vehicle controls validity:

not specified

Negative controls validity:

valid

Remarks:

olive oil

Positive controls validity:

valid

Additional information on results:

the failure to observe any of these effects is compatible with the lack of DNA binding of CS and metabolites, in accordance with (von Daniken et al 1981) and (NTP tr377 report, 1990)

Conclusions:

No genetoxicity (micronucleus) is observed with CS susbtance in mice

Executive summary:

No genetoxicity is observed in micronucleus assay in mice (comparable to OECD 474 methodology) after CS exposure by intraperitoneal and per os routes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

CS substance and its metabolites does not bind to DNA

Justification for classification or non-classification

Some positive results observed in vitro, but negative genotoxicity in vivo. Besides, absence of carcinogenicity is observed in a 2 years inhalation study in vivo.