Trichloroacetyl chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The genotoxicity study on Salmonella typhimurium TA98, TA100 with and without metabolic activation gave negative result.

in vitro chromosomal abberation assay: The neutralized test chemical did not induce chromosomal aberration in human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as genotoxic in vitro.

in vitro mammalian gene mutation assay: The test chemical gave a negative response at one laboratory, with and without S9 and an equivocal (weakly positive) response at a second laboratory in the presence of induced S9.  At the second laboratory, the response was negative without S9. Appropriate responses were obtained with the positive controls indicating the test was sensitive and valid.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Data is from secondary source

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

modified Ames mutagenicity assay in vitro with the histidine-auxotrophic strains

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene

Species / strain / cell type:

S. typhimurium, other: TA98, TA100

Details on mammalian cell type (if applicable):

histidine-auxotrophic strains

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

NA

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

The bacteria were grown overnight in Oxoid nutrient broth, washed and resuspended in 0.1 M phosphate buffer (pH 7.4) to half the original density. The cell suspension, containing about 1 x 10 9 cells/ml, was divided into aliquots of 1.5 ml in centrifuge tubes; then 0.5 ml of either 0.1 M phosphate buffer (pH 7.4) or S9 mix and the test compound (diluted in 10/~1 acetonitrile) was added. The tubes were tightly closed with screw caps and incubated for 90 min at 37°C in a shaking water-bath. The treatment was terminated by centrifugation and resuspension in 1.1 ml fresh buffer. Aliquots of 0.5 ml cell suspension were then added to molten top agar (2 ml each) and duplicate petri dishes containing Vogel-Bonner medium E (= minimal agar) overlaid. For the determination of survival rates, an aliquot of the cell suspension was diluted by a factor of 10(4) in 0.9% NaCI; 10 µl of this dilution was added to 2 ml top agar, containing a 100-fold higher concentration of histidine (5 mM) than that in top agar for counting revertant colonies. Colonies of revertants and survivors were counted after 48 h of incubation at 37°C; the sensitivities of the tester strains were routinely checked with 2- aminoanthracene as a standard mutagen in the presence of S9 mix and with 4-nitro-o-phenylenediamine (TA98) or sodium azide (TA100) in its absence.

Species / strain:

S. typhimurium, other: TA98, TA100

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: other:

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The genotoxicity study on Salmonella typhimurium TA98, TA100 with and without metabolic activation gave negative result.

Executive summary:

Dichloroacetylene (DCA) is a highly reactive compound that decomposes rapidly in contact with air into a series of chlorinated aliphatic hydrocarbons (e.g., phosgene, trichloroacetyl chloride, trichloroacryloyl chloride and hexachlorobutadiene). Experiments were performed to compare the mutagenic properties of DCA and its degradation products on the histidine-dependent tester strains TA98 and TA100 of Salmonella typhimurium. In these experiments, DCA vapour was streamed under analytical control through the bacterial suspensions. DCA is soluble in aqueous solution and was stable under the experimental steady-state conditions of the bacterial exposure. There is a linear correlation between the supply of DCA vapour and solubilized DCA in the range of 1000 and 16000 ppm. Mutagenic response was observed with strain TA100 if the bacteria were suspended in Oxoid medium. No mutagenicity could be detected with strain TA98. DCA mixtures with acetylene, as used as stabilizer for animal experiments, were not mutagenic in either bacterial strain, irrespective of the presence or absence of S9 mix in the cell suspension. One of the degradation products of DCA, trichloroacryloyl chloride, showed pronounced mutagenic properties with and without drug-metabolizing enzymes. Other degradation products of DCA, such as trichloroacetyl chloride and hexachlorobutadiene, were not mutagenic, either in the presence or absence of liver homogenate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

read across substance

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian chromosome aberration was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: 0.5 ml phytohaemagglutinin to 9.0 ml RPMI-1640 tissue culture medium supplemented with 10% fetal bovine serum, 1.0 i.u/ml heparin, 100 i.u/ml penicillin and 100 ug/ml streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9 was prepared from the livers of Alpk rats pretreated with 500 mg Aroclor 1254/kg body weight.

Test concentrations with justification for top dose:

0, 500, 2000 or 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Double deionized water
- Justification for choice of solvent/vehicle: The test chemical was soluble in Double deionized water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Double deionized water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 19 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 10 min

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 cells in metaphase were analysed from each selected
culture for the incidence of chromosomal structural damage according to the criteria of Scott et al. (10) using a X100 oil immersion objective and X10 or X 12.5 eyepieces.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The mitotic index, expressed as a percentage of cells in metaphase, was determined for each culture by analysing 1000 cells using a X25 objective lens and a X10 or X12.5 eyepiece.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for chromosomal aberrations

Statistics:

All slide analyses were recorded directly onto a computerized data storage and retrieval system (ARTEMIS), and the locations of all cells analysed recorded by means of an automatic vernier capture system (Graticules Limited). The total number of cells, excluding those with only gap-type aberrations, for each of the treatment groups was compared with the appropriate solvent control values using the ARTEMIS computer programmed for Fisher's Exact test (one-sided).

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With and without S9, dose related decreases in mean mitotic activity compared with the solvent control were observed and the cultures treated with neutralized test chemical at a concentration of 5000 µg/ml exhibited reduction in mean MI of 51 % & 53% resp

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The highest concentration was based on the limit concentration for this type of assay (5000 µg/ml).

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The neutralized test chemical did not induce chromosomal aberration in human peripheral blood lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as genotoxic in vitro.

Executive summary:

In vitro mammalian chromosome aberration was performed to determine the mutagenic nature of the test chemical. The study was performed usinghuman peripheral blood lymphocytesin the presence and absence of S9 metabolic activation system. The test chemical was dissolved in double distilled water and used at dose level of 0, 500, 2000 or 5000µg/mL. Aliquots of test chemical solutions, sterile double-deionized water or positive controls (cyclophosphamide 50µg/ml, mitomycin Cµg/ml) were administered to duplicate cultures at a volume of 100 uL/10 ml culture. At the same time 200µl of a 1.1 mixture of S9 and co-factor was added to cultures incorporating auxiliary metabolic activation. The cultures were incubated at 37°C for a treatment period of 3 h, centrifuged and the supernatant replaced with

fresh supplemented culture medium containing serum. The cultures were then incubated for a further 19 h at 37°C, treated with colcemid solution (0.4µg/ml) for 2 h and harvested into hypotonic KC1 (0.075 M potassium chloride at room temperature for 10 min) and at least three fixation treatments in 3:1 methanol:glacial acetic acid. Slides were prepared, air dried, stained in a filtered 10% solution of Giemsa stain in double-deionized water buffered to pH 6.8 for 7 min, rinsed in water, air dried and mounted with glass coverslips in DPX. The mitotic index, expressed as a percentage of cells in metaphase, was determined for each culture by analysing 1000 cells using a X25 objective lens and a X10 or X12.5 eyepiece. The appropriate concentrations for chromosomal aberration analysis were selected and the slides coded and

analysed for the presence of chromosomal aberrations. Where possible, 100 cells in metaphase were analysed from each selected culture for the incidence of chromosomal structural damage according to the criteria of Scott et al. using a X100 oil immersion objective and X10 or XI 2.5 eyepieces. The results of the study on neutralized test chemical indicate that no biologically or statistically significant increases in the percentage of aberrant cells (excluding those with only gap type aberrations) were observed following treatment with neutralized test chemical, either in the presence or absence of S9-mix. In both the presence and absence of S9-mix, dose related decreases in mean mitotic activity compared with the solvent control values were observed and the cultures treated with neutralized test chemical at a concentration of 5000 (µg/ml exhibited reductions in mean mitotic index of 51 % and 53% in the absence and presence of S9-mix respectively. In both studies the positive control materials induced statistically and biologically significant increases in the percentage of aberrant cells, thus confirming the metabolic competence of the S9-mix and the ability of the test system to detect chemically induced chromosomal damage. Based on the details of the study,the neutralized test chemical did not induce chromosomal aberration in human peripheral blood lymphocytesin the presence and absence of S9 metabolic activation system and hence it is not likely to classify as genotoxic in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Remarks:

Read across susbstance

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from secondary source

Qualifier:

no guideline followed

Principles of method if other than guideline:

Mouse lymphoma assay was carried out using he given test chemical

GLP compliance:

not specified

Type of assay:

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

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells were maintained at 37 degree C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

No data

Test concentrations with justification for top dose:

0.21, 0.26, 0.33, 0.41, 0.51, and 0.64 uL/mL for the first lab with and without S9, and
0.05, 0.1, 0.2, 0.4, and 0.5 uL/mL for without S9 and 0.0078 to 0.5 uL/mL for with S9 for the second lab

Vehicle / solvent:

dimethylsulfoxide

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: methylcholanthrene +S9

Details on test system and experimental conditions:

Treated cultures contained 6 x 10E6 cells in 10 mL medium. Incubation with test chemical continued for 4 hours, at which time the medium plus chemical was removed, and cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48 hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degree C in 5% carbon dioxide for 10 to 12 days.

Evaluation criteria:

Both responses had to be significant (P<=0.05) for test chemical to be considered capable of inducing TFT resistance. A single significant response led to a questionable conclusion, and the absence of both a trend and a peak response resulted in a negative call.

Statistics:

All data were evaluated statistically for both trend and peak responses.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.64 uL/ml for the first lab and between 0.4 and 0.5 uL/mL for the second lab.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Non mutagenic

Conclusions:

The test chemical gave a negative response at one laboratory, with and without S9 and an equivocal (weakly positive) response at a second laboratory in the presence of
induced S9. At the second laboratory, the response was negative without S9. Appropriate responses were obtained with the positive controls indicating the test was sensitive and valid.

Executive summary:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y TK+/- 3.7.C mouse lymphoma cells in the presence and absence of S9 metabolic activation system at test concentrations of 0.21, 0.26, 0.33, 0.41, 0.51, and 0.64 uL/mL for the first lab with and without S9, and 0.05, 0.1, 0.2, 0.4, and 0.5 uL/mL for without S9 and 0.0078 to 0.5 uL/mL for with S9 for the second lab. The high dose of chemical was determined by solubility and toxicity. L5178Y mouse lymphoma cells were maintained at 37 degree C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels were replicated, including concurrent positive and solvent (dimethylsulfoxide) controls. Ethyl methanesulfonate (without S9) and methylcholanthrene (with S9) were used as positive controls. Treated cultures contained 6 x 10E6 cells in 10 mL medium. Incubation with test chemical continued for 4 hours, at which time the medium plus chemical was removed, and cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48 hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degree C in 5% carbon dioxide for 10 to 12 days. All data were evaluated statistically for both trend and peak responses. Both responses had to be significant (P<=0.05) for test chemical to be considered capable of inducing TFT resistance. A single significant response led to a questionable conclusion, and the absence of both a trend and a peak response resulted in a negative call. Hence, test chemical gave a negative response at one
laboratory, with and without S9 and an equivocal (weakly positive) response at a second laboratory in the presence of induced S9. At the second laboratory, the response was negative without S9. Appropriate responses were obtained with the positive controls indicating the test was sensitive and valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data available for the test chemical was reviewed to determine the mutagenic nature of test chemical, The studies are as mentioned below:

Ames test:

Study 1: Dichloroacetylene (DCA) is a highly reactive compound that decomposes rapidly in contact with air into a series of chlorinated aliphatic hydrocarbons (e.g., phosgene, trichloroacetyl chloride, trichloroacryloyl chloride and hexachlorobutadiene). Experiments were performed to compare the mutagenic properties of DCA and its degradation products on the histidine-dependent tester strains TA98 and TA100 of Salmonella typhimurium. In these experiments, DCA vapour was streamed under analytical control through the bacterial suspensions. DCA is soluble in aqueous solution and was stable under the experimental steady-state conditions of the bacterial exposure. There is a linear correlation between the supply of DCA vapour and solubilized DCA in the range of 1000 and 16000 ppm. Mutagenic response was observed with strain TA100 if the bacteria were suspended in Oxoid medium. No mutagenicity could be detected with strain TA98. DCA mixtures with acetylene, as used as stabilizer for animal experiments, were not mutagenic in either bacterial strain, irrespective of the presence or absence of S9 mix in the cell suspension. One of the degradation products of DCA, trichloroacryloyl chloride, showed pronounced mutagenic properties with and without drug-metabolizing enzymes. Other degradation products of DCA, such as trichloroacetyl chloride and hexachlorobutadiene, were not mutagenic, either in the presence or absence of liver homogenate.

Study 2: Genetic toxicity in vitro study was assessed for test chemical. For this purpose AMES test was performed.The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98 and TA97 in the presence and absence of metabolic activation S9. The S9 mix were prepared from 10% and 30% induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9 repectively. The concentration of test material used in the presence and absence of metabolic activation were 0, 33, 100, 333, 1000, 3333, 10000 µg/plate. No mutagenic effects were observed in any strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be not mutagenic in Salmonella typhimurium TA100, TA1535, TA98 and TA97 by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Study 3: Gene mutation study was performed on Salmonella strains and E.Coli WP2uvrA to determine the mutagenic nature of test chemical. Bacterial strains used were, Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 which have histidine synthesis mutations, the rfa wall mutation, and deletion of the uvrB gene, a tryptophan-dependent E. coli strain WP2uvrA was used in the study. This strain has a uvrA DNA repair deficiency, increasing its sensitivity to detect mutagenic compounds. The study was performed with and without S9 metabolic activation. The S9 mixture contained water, sodium phosphate buffer, glucose 6-phosphate, nicotinamide adenine dinucleotide phosphate (NADP), potassium chloride, magnesium chloride and 10% S9 from Rat liver. Positive control substances used in the experiment included 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, and 4-nitroquinoline-N-oxide. The test concentrations used were 0, 333, 667, 1000, 3330, 5000 μg/plate. Bacteria were prepared on master plates from frozen stocks, inoculated into flasks containing Vogel-Bonner salt solution, supplemented with 2.5% Oxoid nutrient broth No. 2, and harvested for use in late log phase. Strains were confirmed by routine testing for sensitivity to crystal violet (rfa wall mutation) or ampicillin resistance (pKM101 plasmid). Experiments were conducted on two-layer plates. The lower layer was Vogel-Bonner minimal medium E plus 1.5% agar and 0.2% glucose, and the upper layer was 0.7% agar, 0.5% sodium chloride, supplemented with 10 ml of 0.5 mM histidine/ biotin, or 0.5 mM tryptophan, per 100 ml of agar when appropriate (Vogel and Bonner, 1956). All tests were conducted in triplicate. Aliquots of the culture were used to quantify the number of spontaneous revertants for both bacterial species. Background lawn was evaluated for evidence of cytotoxicity. Revertants colonies were counted manually, or using an automated colony counters. Dose-ranging studies demonstrated no cytotoxic effects of test chemical up to 5 mg per plate using Salmonella strains TA100 and WP2uvrA. Numbers of revertant colonies were present in the historical range with and without metabolic activation. Therefore, the test chemical can be considered as not mutagenic to Salmonella typhimurium Strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA.

in vitro chromosomal abberation assay:

Study 1: In vitro mammalian chromosome aberration was performed to determine the mutagenic nature of the test chemical. The study was performed usinghuman peripheral blood lymphocytesin the presence and absence of S9 metabolic activation system. The test chemical was dissolved in double distilled water and used at dose level of 0, 500, 2000 or 5000µg/mL. Aliquots of test chemical solutions, sterile double-deionized water or positive controls (cyclophosphamide 50µg/ml, mitomycin Cµg/ml) were administered to duplicate cultures at a volume of 100 uL/10 ml culture. At the same time 200µl of a 1.1 mixture of S9 and co-factor was added to cultures incorporating auxiliary metabolic activation. The cultures were incubated at 37°C for a treatment period of 3 h, centrifuged and the supernatant replaced with fresh supplemented culture medium containing serum. The cultures were then incubated for a further 19 h at 37°C, treated with colcemid solution (0.4µg/ml) for 2 h and harvested into hypotonic KC1 (0.075 M potassium chloride at room temperature for 10 min) and at least three fixation treatments in 3:1 methanol:glacial acetic acid. Slides were prepared, air dried, stained in a filtered 10% solution of Giemsa stain in double-deionized water buffered to pH 6.8 for 7 min, rinsed in water, air dried and mounted with glass coverslips in DPX. The mitotic index, expressed as a percentage of cells in metaphase, was determined for each culture by analysing 1000 cells using a X25 objective lens and a X10 or X12.5 eyepiece. The appropriate concentrations for chromosomal aberration analysis were selected and the slides coded and analysed for the presence of chromosomal aberrations. Where possible, 100 cells in metaphase were analysed from each selected culture for the incidence of chromosomal structural damage according to the criteria of Scott et al. using a X100 oil immersion objective and X10 or XI 2.5 eyepieces. The results of the study on neutralized test chemical indicate that no biologically or statistically significant increases in the percentage of aberrant cells (excluding those with only gap type aberrations) were observed following treatment with neutralized test chemical, either in the presence or absence of S9-mix. In both the presence and absence of S9-mix, dose related decreases in mean mitotic activity compared with the solvent control values were observed and the cultures treated with neutralized test chemical at a concentration of 5000 (µg/ml exhibited reductions in mean mitotic index of 51 % and 53% in the absence and presence of S9-mix respectively. In both studies the positive control materials induced statistically and biologically significant increases in the percentage of aberrant cells, thus confirming the metabolic competence of the S9-mix and the ability of the test system to detect chemically induced chromosomal damage. Based on the details of the study,the neutralized test chemical did not induce chromosomal aberration in human peripheral blood lymphocytesin the presence and absence of S9 metabolic activation system and hence it is not likely to classify as genotoxic in vitro.

Study 2: In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed usingCHL cells both in the presence and absence of S9 metabolic activation system. The test chemical was studies at dose level of 40-100% (concentration in air) with 6 hrs treatment and 18 hrs recovery. The test chemical did not induce chromosomal aberration in CHL cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

in vitro mammalian gene mutation assay:

Study 1: In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y TK+/- 3.7.C mouse lymphoma cells in the presence and absence of S9 metabolic activation system at test concentrations of 0.21, 0.26, 0.33, 0.41, 0.51, and 0.64 uL/mL for the first lab with and without S9, and 0.05, 0.1, 0.2, 0.4, and 0.5 uL/mL for without S9 and 0.0078 to 0.5 uL/mL for with S9 for the second lab. The high dose of chemical was determined by solubility and toxicity. L5178Y mouse lymphoma cells were maintained at 37 degree C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. All treatment levels were replicated, including concurrent positive and solvent (dimethylsulfoxide) controls. Ethyl methanesulfonate (without S9) and methylcholanthrene (with S9) were used as positive controls. Treated cultures contained 6 x 10E6 cells in 10 mL medium. Incubation with test chemical continued for 4 hours, at which time the medium plus chemical was removed, and cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48 hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 degree C in 5% carbon dioxide for 10 to 12 days. All data were evaluated statistically for both trend and peak responses. Both responses had to be significant (P<=0.05) for test chemical to be considered capable of inducing TFT resistance. A single significant response led to a questionable conclusion, and the absence of both a trend and a peak response resulted in a negative call. Hence, test chemical gave a negative response at one laboratory, with and without S9 and an equivocal (weakly positive) response at a second laboratory in the presence of induced S9. At the second laboratory, the response was negative without S9. Appropriate responses were obtained with the positive controls indicating the test was sensitive and valid.

Study 2: In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y TK+/- 3.7.C mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The mouse lymphoma cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68. The test chemical was dissolved in DMSO and the cells at a concentration of 1.2 ×10⁷/mL were exposed for 4 h to a range of concentrations from 0.5-30 µg/ml. Concurrent solvent and positive control chemicals were also included in the study. The cells were washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 X 10⁵/mL at 24 h intervals. They were then cloned 1 X 10⁶cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar. Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 g/mL) to the cloning medium for mutant selection. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter. Only colonies larger than approx. 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10⁶surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The test chemical was considered to have no mutagenic inducing potency on the mammalian cell line in presence and absence of S9 metabolic activation system.

Based on the observations made, the test chemical does not exhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the observations made, the test chemical does not exhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.