Registration Dossier
Registration Dossier
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EC number: 900-600-0 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A number of mutagenicity studies are available on the three constituents of Flux1. In this case one study has been selected for each constituent and each endpoint as the most representative and most relevant, and are summarised below. Overall, based on the data for the constituents, Flux1 is anticipated to provide positive results for mutagenicity in bacteria and mammalian cells, but negative results for cytogenicity.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: - Guideline study (OECD, etc.) - number of replicates to low for some concentrations
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- : gas exposure method
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- no data
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Species / strain / cell type:
- E. coli WP2
- Additional strain / cell type characteristics:
- other: carrying the plasmid pKM101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 extract from phenobarbitone + 5,6-benzoflavone treated male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- 100, 500, 1000, 2000, 5000, 10'000, 20'000, 50'000 ppm (= 640, 3200, 6400, 12'800, 32'000, 64'000, 128'000, 320'000 mg/m³)
assuming 500 mL air volume/plate: 0.8 - 160 mg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: none
- Untreated negative controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamidMe, MMC: mitomycin C, 2AA: 2-aminoanthracen, see table 1 for details
- Remarks:
- with the gas exposure, sham exposed controls are both negative and true negative controls
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: gas exposure method
DURATION
- Preincubation period: 0 min, no preincubation
- Exposure duration: 24 h
- Expression time (cells in growth medium): 0 h
- Selection time (if incubation with a selection agent): 24 h (+24 h during exposure)
SELECTION AGENT (mutation assays): Salmonella strains: L-histidine, Ecoli strains: L-trytophane
DETERMINATION OF CYTOTOXICITY
- Method: not reported
- Evaluation criteria:
- - a factor of 2 between the average no of revertants on exposed plates compared to the everage no of revertants on unexposed plates
- Statistics:
- - Bartlett's test for evaluation of homogenity of variance,
- one way ANOVA in the case of variance homogenity
- Kruskal-Wallis- rank sum test for non homogenous variance - Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- only at concentrations > 5000 ppm
- Cytotoxicity / choice of top concentrations:
- other: only at the highest concentration
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- only at concentrations > 5000 ppm
- Cytotoxicity / choice of top concentrations:
- other: only at the highest concentration
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- without activation: only at concentrations > 500 ppm, with activation: only at concentrations > 2000 ppm
- Cytotoxicity / choice of top concentrations:
- other: only at the highest concentration
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not expected as CTC is apolarm and stable
- Effects of osmolality: not expected as CTC is apolarm and stable
- Evaporation from medium: not applicable due to gas exposure method
- Water solubility: not applicable due to gas exposure method
- Precipitation: not applicable due to gas exposure method
- Other confounding effects: not expected
RANGE-FINDING/SCREENING STUDIES: No
COMPARISON WITH HISTORICAL CONTROL DATA: No
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The maximum exposure concentration was 2% or 5%; carbon tetrachloride showed toxicity for all strains in tests conducted with 5% of the agents
see tables I - II for detailed results - Remarks on result:
- other: strain/cell type: TA 98
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
ambiguous
- A CTC concentration dependent rise of the no. of revertants was found in the strains TA98, E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101
- The used concentrations are very high
- Ambiguous results - Executive summary:
The study Araki (2004) presents an ambigous result concerning genotoxicity of CTC in the bacterial reverse mutation assay.
Salmonella strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli strains E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101 were exposed to CTC using the gas exposure method.
A CTC concentration dependent rise of the no. of revertants was found in the strains TA98, E. coli WP2 uvr A pKM 101 and E. coli WP2 pKM 101. But for TA98, E. coli WP2 uvr A pKM 101 the concentrations where the criterion of 2 fold increase of no. of revertants compared to controls were reached were 10´000 ppm. And the results for E. coli WP2 pKM 101 (criterion reached at 1000 ppm) were not reproduced and based only on 2 replicate plates per concentration.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- dossier on the toxicological profile of CTC
- Type of information:
- other: dossier on the toxicological profile of CTC
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: secondary source
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Summay of the toxicological profile of CTC
- Type of assay:
- other: summary of the toxicological profile of CTC
- Remarks on result:
- other: See 'Overall remarks'
- Conclusions:
- The summary of in vitro genotoxicity tests for CTC as laid down in the ATDSR dossier is ambiguous and does not yield a definite classification of CTC for this endpoint. The results suggest a possible mechanism of metabolic activation.
- Executive summary:
Ames-Test are mainly negative, in reliable studies only strains sensible for oxidative damage give positive results as do most tests with Aspergillus nidulans and Saccharomyces cerevisiae. The effects seen in the S. cerevisiae tests might be due to double strand breaks induced by oxidative damage.
Results from tests with mammalian cell lines give both possitive and negative results using different techniques, the negative results being the majority. The experiments of Doherty 1996 suppose that metabolic activation of CTC is needed to develop its genotoxic potential.
This is also supported by studies analysis the DNA-adduct formation induced by CTC. Both direct (CTC metabolites binding to DNA) as well as indirect (DNA-adducts indicative for oxidative stress) effects were found in different experiments.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1981
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study, sufficient documentation.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- The hgprt locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 20, 30, 40, 50 mM
- Vehicle / solvent:
- Vehicle: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- EMS was used as a positive control in the presence of a metabolic activating system (S9)
- Positive control substance:
- other: N - nitrosodiumethylamine (DMN)
- Remarks:
- DMN was used as a positive control in the absence of a metabolic activating system (S9)
- Details on test system and experimental conditions:
- Cells were subcultured on days 1, 3 and 6 after mutagen treatment and selected for 6-TG resistance on day 8. All experiments were performed under gold light to minimise mutagenic effects mediated by cool white light.
- Evaluation criteria:
- A compound was considered to exhibit a mutagenic response if the slope of mutation induction as a function of test concentrations was greater than the control at the 99 % significance level according to the t-test.
- Statistics:
- See above
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- other:
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- 1,2-dichloroethane exhibited mutagenic activity, expressed as mutation frequency (mutants /10 E06 clonable cells) to 1,2-dichloroethane concentration), either in the absence or the presence of a metabolic activation system (since the slope of the straight line fit of mutation induction was different from the control at > t0.99) in a concentration-dependent manner.
Mutagenic activity was increased approx. 4-fold in the presence of the metabolic activating system compared to the results in the absence of a metabolic activating system.
Regarding cytotoxicity, in the absence of S9-mix, cell survival was reduced to 50 % at approx. 50 mM. The cytotoxic effect was even more pronounced in the presence of S9-mix. - Remarks on result:
- other: strain/cell type: Chinese hamster ovary (CHO) cells
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation
Under the study conditions, 1,2-dichloroethane showed a mutagenic effect in a mammalian cell gene mutation assay in the CHO/HGPRT system. - Executive summary:
1,2 -dichloroethane, in concentrations of 0.05–50 mM, was tested for its mutagenic activity in the Chinese hamster ovary cell / hypoxanthine - guanine phosphoribosyl transferase (CHO / HGPRT) system in the absence and presence of a metabolic activation system (S9 -mix). Ethyl methanesulphonate (EMS) and dimethylnitrosoamine (DMN) were used as positive controls in the absence and presence of a metabolic activation, respectively. DMSO was used as a negative/vehicle control. Cells were subcultured on days 1, 3 and 6 after mutagen treatment and selected for 6 -TG resistance on day 8. All experiments were performed under gold light to minimise mutagenic effects mediated by cool white light. A compound was considered to exhibit a mutagenic response if the slope of mutation induction as a function of test concentrations was greater than the control at the 99 % significance level (t0.99) according to the t-test.
1,2 -dichloroethane exhibited mutagenic activity, expressed as mutation frequency (mutants/10 E06 clonable cells) to 1,2 -dichloroethane concentration), either in the absence or the presence of a metabolic activation system in a concentration-dependent manner. Mutagenic activity was increased approx. 4-fold in the presence of metabolic activating system compared to the results obtained in the absence of a metabolic activating system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Meets generally accepted scientific standards, well-documented and acceptable for assessment.
- Principles of method if other than guideline:
- The test substance was tested in the strains TA 1535, TA 100, TA 1537 and TA 98 in a standard plate test or in the strains TA 1535 and TA 100 in a modified exsiccator test for it's mutagenic potential.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- other: S. typhimurium TA 1535 and TA 100 (exsiccator test)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix
- Test concentrations with justification for top dose:
- 20 - 5000 µg/plate standard plate test, 3000 and 6000 µl/30 l in an exsiccator (modified test)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: The test substance was completely soluble in DMSO - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with S-9 mix for strains TA 100, TA 98, TA 1535 and TA 1537
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: N-Methyl-N'-nitro-N-nitroso-guanidine
- Remarks:
- without S-9 mix for strain TA 100 and TA 1535
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-o-phenylendiamine
- Remarks:
- without S-9 mix for strain TA 98
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 9-aminoacridine chloride
- Remarks:
- without S-9 mix for stain TA 1537
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) and in a modified exsiccator test
DURATION
- Exposure duration: ca. 48 hours at 37 °C (plate incorporation); 4 hours at 37°C in the exsiccator and then 44 hours at 37°C
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results. - Statistics:
- no data
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive - Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study, linguistic limitations.
- Principles of method if other than guideline:
- Method: other: no data
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Chinese hamster lung (CHL) fibroblasts
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- 0, 500, 1000, 2000, 4000, 6000 µg/ml (see: Method)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks on result:
- other: other: Chinese hamster lung (CHL) fibroblasts
- Remarks:
- Migrated from field 'Test system'.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- GLP guideline study according to the OECD Guideline No 476. The study has been well recognised by official reviews and was quoted with Klimisch score 1 in the European Risk Assessment Report for chloroform (France 2007). However, the original study is not published.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- HPRT-gene
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- 6-thioguanine resistant mutants
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Without S9: 100, 500, 1000 and 1500 µg/ml
With S9: 100, 500, 1000, 1250 (only in experiments 2 and 3) and 1500 µg/ml. - Vehicle / solvent:
- Information not available
- 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:
- Chloroform was examined for induction of 6-thioguanine resistant mutants in V79 Chinese hamster cells (HGPRT-test) in the presence and absence of a fraction of rat liver homogenate for metabolic activation. A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance did not produce any significant cytotoxic effect with and without metabolic activation up to the limit of solubility (1500 µg/ml). For mutagenicity testing three independent experiments with metabolic activation and 2 without metabolic activation were performed. In the absence of S9, the dose levels were 100, 500, 1000 and 1500 µg/ml; in the presence of S9, dose levels were 100, 500, 1000, 1250 (only in experiments 2 and 3) and 1500 µg/ml. EMS (without S9) and DMBA (with S9) were used as positive controls.
- Evaluation criteria:
- Information not available
- Statistics:
- Information not available
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- other: > 1500 µg/ml
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Additional information on results:
- No relevant cytotoxicity was observed in the main experiments. Without S9, chloroform did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level. Inconclusive with S9-mix in the dose range of 1000 up to 1500 µg/ml; slight increases in mutant rates in 2/3 experiments with generally very pronounced variations of the gene mutation rates; maximum mutation rate 56.2 x 10-6 (negative control 31.9 x 10-6). Marked increases in the mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
ambiguous with metabolic activation
negative without metabolic activation - Executive summary:
Chloroform was examined for induction of 6-thioguanine resistant mutants in V79 Chinese hamster cells (HGPRT-test) in the presence and absence of a metabolic activation. Without S9, chloroform up to 1500 µl/ml did not induce a significant increase of the mutant colonies or in the mutation frequency. With S9, ambigous results (slight increases in mutant rates in 2/3 experiments) were observed. No relevant cytotoxicity was observed in the main experiments.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: The study mainly followed the principles laid down in the OECD Guideline for Testing of Chemicals No. 471.
- Principles of method if other than guideline:
- A gas-phase exposure method using a gas sampling bag (Araki, A., Noguchi, T., Kato, F., and Matsushita, T. 1994. Mutat. Res. 307, 335-344) was applied.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix and glutathione-supplemented s9 mix
- Test concentrations with justification for top dose:
- 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 % chloroform in gas phase
- Vehicle / solvent:
- no vehicle/solvent
- Untreated negative controls:
- yes
- Remarks:
- air
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Positive controls:
- yes
- Positive control substance:
- other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- Gas-phase exposure using a gas sampling bag [Araki et al., 1994] was conducted with slight modifications. A (l.5 ml. aliquot of S9* mix (for assays with metabolic activation) or 0.1 M sodium phosphate buffer (pH 7.4: for assays without metabolic activation) plus 0.1 mL of tester strain. which had been cultured at 37°C for 10 h in nutrient broth, were mixed with 2 mL of molten top agar containing 0.05 µmol/ml of L-histidine and D-biotin (for tests conducted with Salmonella) or 0.05 µmol/ml_ of L-tryptophan (for tests conducted with E. coli), and immediately poured into plates containing 30 mL of Vogel-Bonner minimal glucose agar (Oriental Yeast). The plates were kept on a flat table until the top agar solidified. The plates then were placed separately, upside down without their lids, in a plate holder, arranging the plates according to their level of exposure and the activation system used. The plate holder was placed in a 10L gas exposure bags through an opening made by scissors in one side of the bag. The bag was then closed by folding the open side 3 times and sealing with adhesive tape. The exposure concentrations used in the test were calculated from the volumes of the test compound vapor and air diluent in the gas exposure bag at the exposure temperature. First, the air trapped in the exposure bag after loading the plate holder was removed using a pump. A fixed volume of diluent air to produce a final exposure volume of 500 mL per plate then was pumped into the gas exposure bag. The test compound was injected through a septum directly into the gas exposure bag using a syringe. and the solution in the bag was vaporized by placing the portion of the bag making contact with the test agent on a hot-plate (at 50-60°C). After vaporization, the bag was squeezed by hand, to mix the test compound vapor and air thoroughly. The bacterial plates in the gas exposure bag were incubated at 37°C for 24 h. After the 24 h exposure, the test compound vapor was removed from the gas exposure bag. The seal on the gas exposure bag was peeled off, and the plate holder was taken out of the bag. The plates were held in a safety cabinet for 10 min in order to remove the test compound, and then the lids of the plate were replaced. The plates with lids were turned upside down. transferred separately to a vinyl bag, according to their concentration level and whether with/without the S9 mix, and incubated an additional 24 h at 37°C. The maximum exposure concentration was 5%. Chloroform showed toxicity for all strains in tests conducted with 5%. The sensitivity of tester strains and the activity of the S9 mix were established by testing positive control substances by the pourplate method. Two plates per dose were employed in assays conducted with the test agents: negative control assays (exposure to airs used 4 plates. *S9 was a liver homogenate fraction prepared from a male Sprague-Dawley rat pretreated with sodium phenobarbital and 5,6-benzoflavone.
- Evaluation criteria:
- The twofold rule (Ames et al. 1975) was used for determining mutagenicity in individual experiments.
- Species / strain:
- E. coli, other: WP2uvrA/pKM101, WP2/pKM101
- Metabolic activation:
- with and without
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- other: 5 % in gas phase
- Additional information on results:
- Chloroform concentrations of 0.5-2 % produced weakly dose-dependent increases in revertant colonies of more than twice that of the negative control in WP2/pKM101, but only in the presence of glutathione-supplemented S9 mix. The induction of revertant colonies was statistically significant, but on average, the number of revertant colonies was increased only 2.1-fold in WP2/pKM101. Both lots of the test agent, APJ7886 and CG01, produced similar mutagenic responses in WP2/pKM101. Chloroform was not mutagenic in WPuvrA/pKM101, with or without S9 mix, and no mutagenicity was observed in WPuvrA/pKM101 in the presence of glutathione-supplemented S9 mix.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Executive summary:
The present study investigated the mutagenicity of chloroform in E. coli WP2 uvr A pKM 101 using a gas exposure method. Mutagenicity was tested with and without a metabolic activation system and additionally with a glutathione-supplemented metabolic activation system.
The tester strains were exposed for 24 hours to chloroform concentrations in the gas phase of 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 % at 37°C. The sensitivity of tester strains and the activity of the S9 mix were established by testing positive control substances by the pour-plate method. Two plates per dose were employed in assays conducted with the test agents. Negative control assays (exposure to air) used four plates.
The maximum exposure concentration of 5 % chloroform in the gas phase was toxic for all strains. Chloroform was not mutagenic in E. coli WP2 uvr A pKM 101 , with or without S9 mix, as in the presence of glutathione-supplemented S9 mix.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- This study has several shortcomings. First, the positive control was not concurrent. Second, there is no indication that evaporation and escape of CHCl3 was prevented. Third, the maximal dose was only 400 µg/ml. Fourth, information about the toxicity of CHCl3 for the lymphocytes was not provided.
- Principles of method if other than guideline:
- Method: other: Thomson & Evans (1979) Mut. Res., 67, 47
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Human lymphocytes
- Metabolic activation:
- with
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 50, 100, 200 and 400 µg/ml
- Details on test system and experimental conditions:
- Peripheral blood from a young, healthy volunteer was cultured at 37°C for 24 hr, duplicate cultures were then treated with 0.1-ml aliquots of chloroform in acetone to give final concentrations of 50, 100, 200 and 400 µg/ml and 0.1 ml 10% S-9 mix was added to each culture. The treated cultures were incubated for 2 hr at 37°C. At the end of the treatment the cells were centrifuged, resuspended in fresh medium without PHA, and reincubated for a further 22 hr to bring first cycle cells to mitosis after a total of 48 hr culture. One hour before harvest, colcemid was added to each culture. Cells were collected and fixed, and slides prepared, stained with Giemsa and mounted using conventional methods. Negative controls were cultures treated with 0.1 ml acetone and 0.1 ml S.9 mix. Positive controls were not included since the donor's lymphocytes had previously shown dose-related chromosome breakage after treatment with benzo[a]pyrene in the presence of S.9 mix. Slides were coded by an independent observer, and 100 well spread metaphases with 46 or more chromosomes were scored from each culture, making a total of 200 cells per treatment.
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: >400 µg/ml
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not valid
- Additional information on results:
- The response of this donor's lymphocyte chromosomes to CHCl3 was a random variation around the control value. The highest breakage level was at 200 µg/ml with 8 breaks/100 cells compared with 5.5 breaks/100 cells in the control. This difference was not significant in a chi-square test.
- Remarks on result:
- other: other: Human lymphocytes
- Remarks:
- Migrated from field 'Test system'.
- Executive summary:
The potential of chloroform to induce structural chromosome aberrations in human lymphocytes was evaluated with metabolic activation system at the dose-levels of 50, 100, 200 and 400 µg/ml . Cytotoxicity was noted at dose-levels higher than 400 µg/ml. Under these experimental conditions, the test item did not induce any noteworthy increase in the number of cells with structural chromosome aberration with S9 mix, however, there were no indication that evaporation and escape of CHCl3 was prevented. In adition, the positive control was not concurrent and the maximal dose was only 400 µg/ml.
Referenceopen allclose all
- Table Ia Mutagenicity Testing of Carbon Tetrachloride in S.typhimurium TA98
TA98 |
||||||||||
Lot. Nr. APE7331 |
Lot No. KSG7670 |
|||||||||
Dose % |
Test 1 (Mean) |
Test 2 (Mean) |
Test 3 (Mean |
Test 1-3 Mean + SD |
||||||
Negative control (Air) |
9 |
16 |
13 |
18 |
8 |
15 |
||||
13 |
16 |
(14) |
17 |
13 |
(15) |
9 |
13 |
(11) |
13+3.3 |
|
Without S 9 mix |
||||||||||
0.005 |
NT |
NT |
13 |
23 |
(18) |
NT |
NT |
18a |
||
0.01 |
NT |
NT |
16 |
20 |
(18) |
NT |
NT |
18a |
||
0.05 |
NT |
NT |
28 |
16 |
(22) |
11 |
16 |
(14) |
18+7.2 |
|
0.1 |
13 |
20 |
(17) |
22 |
23 |
(23) |
11 |
13 |
(12) |
17+5.3 |
0.2 |
NT |
NT |
NT |
NT |
15 |
17 |
(16) |
16a |
||
0.5 |
29 |
20 |
(25) |
28 |
31 |
(30) |
10 |
22 |
(16) |
23+7.8* |
1 |
40 |
24 |
(32) |
21 |
21 |
(21) |
26 |
21 |
(24) |
26+7.4** |
2 |
NT |
NT |
NT |
NT |
32 |
36 |
(34) |
34a |
||
5 |
36 |
38 |
(37) |
34 |
30 |
(32) |
31 |
17 |
(24) |
31+7.5 |
AF2 0.1 μg/plate |
719 |
680 |
(700) |
627 |
614 |
(621) |
584 |
596 |
(590) |
|
AF2 0.005 μg/plate |
||||||||||
TA98 |
||||||||||
Lot. Nr. APE7331 |
Lot No. KSG7670 |
|||||||||
Dose % |
Test 1 (Mean) |
Test 2 (Mean) |
Test 3 (Mean |
Test 1-3 Mean + SD |
||||||
Negative control (Air) |
22 |
28 |
26 |
39 |
14 |
28 |
||||
24 |
26 |
(25) |
36 |
32 |
(33) |
24 |
23 |
(22) |
27+6.6 |
|
With S 9 mix |
||||||||||
0.005 |
NT |
NT |
37 |
29 |
(33) |
NT |
NT |
33a |
||
0.01 |
NT |
NT |
32 |
26 |
(29) |
NT |
NT |
29a |
||
0.05 |
NT |
NT |
22 |
32 |
(27) |
11 |
23 |
(17) |
22+8.6 |
|
0.1 |
29 |
30 |
(30) |
37 |
22 |
(30) |
29 |
23 |
(26) |
28+5.4 |
0.2 |
NT |
NT |
NT |
NT |
21 |
26 |
(24) |
24a |
||
0.5 |
25 |
26 |
(26) |
25 |
28 |
(25) |
28 |
28 |
(28) |
26+1.5 |
1 |
33 |
36 |
(35) |
31 |
38 |
(35) |
31 |
24 |
(28) |
32+4.9 |
2 |
NT |
NT |
NT |
NT |
36 |
32 |
(34) |
34a |
||
5 |
52 |
38 |
(45) |
30 |
36 |
(33) |
24 |
28 |
(26) |
35+9.9 |
2AA 0.5 μg/plate |
332 |
355 |
(344) |
343 |
450 |
(397) |
444 |
436 |
(440) |
|
2AA 2.0 μg/plate |
||||||||||
* P<0.05
** P<0.01, compared with results of negative control group in each strain
NT not ested
AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
2AA 2-aminoanthracene
a Statistical analysis was not applied because number of measurements was less than three
- Table Ib Mutagenicity Testing of Carbon Tetrachloride in E.coli WP2uvrA/pKM101
TA98 |
||||||||||
Lot. Nr. APE7331 |
Lot No. KSG7670 |
|||||||||
Dose % |
Test 1 (Mean) |
Test 2 (Mean) |
Test 3 (Mean |
Test 1-3 Mean + SD |
||||||
Negative control (Air) |
9 |
16 |
13 |
18 |
8 |
15 |
||||
13 |
16 |
(14) |
17 |
13 |
(15) |
9 |
13 |
(11) |
13+3.3 |
|
Without S 9 mix |
||||||||||
0.005 |
NT |
NT |
13 |
23 |
(18) |
NT |
NT |
18a |
||
0.01 |
NT |
NT |
16 |
20 |
(18) |
NT |
NT |
18a |
||
0.05 |
NT |
NT |
28 |
16 |
(22) |
11 |
16 |
(14) |
18+7.2 |
|
0.1 |
13 |
20 |
(17) |
22 |
23 |
(23) |
11 |
13 |
(12) |
17+5.3 |
0.2 |
NT |
NT |
NT |
NT |
15 |
17 |
(16) |
16a |
||
0.5 |
29 |
20 |
(25) |
28 |
31 |
(30) |
10 |
22 |
(16) |
23+7.8* |
1 |
40 |
24 |
(32) |
21 |
21 |
(21) |
26 |
21 |
(24) |
26+7.4** |
2 |
NT |
NT |
NT |
NT |
32 |
36 |
(34) |
34a |
||
5 |
36 |
38 |
(37) |
34 |
30 |
(32) |
31 |
17 |
(24) |
31+7.5 |
AF2 0.1 μg/plate |
719 |
680 |
(700) |
627 |
614 |
(621) |
584 |
596 |
(590) |
|
AF2 0.005 μg/plate |
||||||||||
TA98 |
||||||||||
Lot. Nr. APE7331 |
Lot No. KSG7670 |
|||||||||
Dose % |
Test 1 (Mean) |
Test 2 (Mean) |
Test 3 (Mean |
Test 1-3 Mean + SD |
||||||
Negative control (Air) |
22 |
28 |
26 |
39 |
14 |
28 |
||||
24 |
26 |
(25) |
36 |
32 |
(33) |
24 |
23 |
(22) |
27+6.6 |
|
With S 9 mix |
||||||||||
0.005 |
NT |
NT |
37 |
29 |
(33) |
NT |
NT |
33a |
||
0.01 |
NT |
NT |
32 |
26 |
(29) |
NT |
NT |
29a |
||
0.05 |
NT |
NT |
22 |
32 |
(27) |
11 |
23 |
(17) |
22+8.6 |
|
0.1 |
29 |
30 |
(30) |
37 |
22 |
(30) |
29 |
23 |
(26) |
28+5.4 |
0.2 |
NT |
NT |
NT |
NT |
21 |
26 |
(24) |
24a |
||
0.5 |
25 |
26 |
(26) |
25 |
28 |
(25) |
28 |
28 |
(28) |
26+1.5 |
1 |
33 |
36 |
(35) |
31 |
38 |
(35) |
31 |
24 |
(28) |
32+4.9 |
2 |
NT |
NT |
NT |
NT |
36 |
32 |
(34) |
34a |
||
5 |
52 |
38 |
(45) |
30 |
36 |
(33) |
24 |
28 |
(26) |
35+9.9 |
2AA 0.5 μg/plate |
332 |
355 |
(344) |
343 |
450 |
(397) |
444 |
436 |
(440) |
|
2AA 2.0 μg/plate |
||||||||||
* P<0.05
** P<0.01, compared with results of negative control group in each strain
NT not ested
AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
2AA 2-aminoanthracene
a Statistical analysis was not applied because number of measurements was less than three
- Table II Mutagenicity Testing of Carbon Tetrachloride and chloroform in E.coli WP2/pKM101
Carbon Tetrachloride Lot No. APE7331 |
|||
Dose (%) |
Mean |
||
Negative control (Air) |
20 |
37 |
|
29 |
38 |
(30) |
|
Without S9 mix |
|||
0.01 |
44 |
43 |
(44)* |
0.05 |
54 |
61 |
(58)* |
0.1 |
74 |
83 |
(79)* |
0.2 |
NT |
NT |
|
0.5 |
138 |
155 |
(147)* |
1 |
243 |
230 |
(237)* |
2 |
320 |
367 |
(344)* |
5 |
232 |
208 |
(12)* |
AF2 0.1μg/plate 0.1μg/plate |
460 |
456 |
(220)* |
Negative Control (Air) |
48 |
71 |
|
53 |
51 |
(56) |
|
With S9 mix |
|||
0.01 |
55 |
59 |
(57)* |
0.05 |
70 |
39 |
(55)* |
0.1 |
91 |
85 |
(88)* |
0.2 |
NT |
NT |
|
0.5 |
150 |
180 |
(165)* |
1 |
288 |
284 |
(286)* |
2 |
445 |
395 |
(420)* |
5 |
101 |
71 |
(86)* |
2AA 10μg/plate |
272 |
358 |
(315) |
* P<0.05
** P<0.01, compared with results of negative control group in each strain
NT not ested
AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
2AA 2-aminoanthracene
a Statistical analysis was not applied because number of measurements was less than three
No remarks
Standard plate test
Without S-9 mix: a slight increase of the mutation rate with TA 1535 at 2500 and 5000 µg (factor 1.7); no increase was observed with TA 100, TA 1537 and TA 98.
With S-9 mix: a slight increase of the mutation rate with TA 1535 in a dose dependent manner reaching factor 2 at a dose of 5000 µg; no increase was observed with TA 100, TA 1537 and TA 98.
Modified exsiccator test:
Without S-9 mix: an increase of factor 1.7 to 4.7 with TA 1535 (3000 µl/30 l exsiccator), increase of factor 4.3 to 5.3 with TA 1535 (6000 µl/30 l exsiccator), only slight increase (factor 1.7) with TA 100 in 1/2 experiments (6000 µl/30 l exsiccator).
With S-9 mix (without glutathione): an increase of factor 6.1 to 8.9 with TA 1535 (3000 µl/30 l exsiccator), increase of factor 5.8 to 10.0 with TA 1535 (6000 µl/30 l exsiccator), only slight increase (factor 2.2) with TA 100 in 1 out of 2 experiments (3000 µl/30 l exsiccator), only slight increase (factor 1.8 -2.3) with TA 100 (6000 µl/30 l exsiccator) with S-9 mix (supplemented with glutathione): mutagenic with TA 1535 at both concentrations (factor 30.5 -41.2), increase of factor 2.0 to 4.9 with TA 100 depending on the amount of test substance.
Without S9-mix, no increases in chromosomal aberrations up
to 4 mg/ml following 24- and 48-h exposure, ambiguously positive at 6000
µg/ml. With S9-mix, a dose-related increase in chromosomal effects was
noted at 1000 and 2000 µg/ml. No effect at 500 µg/ml:
4000 µg/l was cytotoxic (no mitosis).
The structural abnormalities comprised primarily significant
increases in chromatid breaks and chromatid exchanges, but
no chromosomal breaks or exchanges.
Table 1: Mutagenicity testing of chloroform in E. coli WP2/pKM101
-- |
Chloroform |
|||||||||
-- |
Lot no. APJ7886 |
Lot no. CG01 |
||||||||
Dose (%) |
Test 1 (Mean) |
Test 2 (Mean) |
Test 3 (Mean) |
Test 1-3 (Mean +/- SD) |
||||||
Negative control (air) |
20 |
37 |
-- |
68 |
63 |
-- |
48 |
59 |
-- |
-- |
29 |
34 |
(30) |
62 |
47 |
(60) |
52 |
54 |
(53) |
48 ± 15 |
|
Without S9 mix |
||||||||||
0.01 |
38 |
28 |
(33) |
NT |
NT |
-- |
NT |
NT |
-- |
33 a) |
0.05 |
31 |
30 |
(31) |
55 |
55 |
(55) |
47 |
54 |
(51) |
45 ± 12 |
0.1 |
36 |
40 |
(38) |
52 |
72 |
(62) |
56 |
53 |
(55) |
52 ± 13 |
0.2 |
NT |
NT |
53 |
49 |
(51) |
51 |
46 |
(49) |
50 ± 3 |
|
0.5 |
37 |
31 |
(34) |
46 |
46 |
(46) |
38 |
45 |
(42) |
41 ± 6.2 |
1 |
44 |
33 |
(39) |
81 |
60 |
(71) |
49 |
61 |
(55) |
55 ± 17 |
2 |
46 |
36 |
(41) |
49 |
52 |
(56) |
47 |
38 |
(43) |
46 ± 8.6 |
5 |
10 |
9 |
(10) |
17 |
31 |
(24) |
24 |
28 |
(26) |
20 ± 9.3 |
AF2 0.1 microg/plate |
460 |
456 |
(458) |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MMC 0.1 microg/plate |
-- |
-- |
-- |
247 |
250 |
(249) |
227 |
233 |
(230) |
-- |
Negative control (air) |
48 |
71 |
-- |
67 |
71 |
-- |
53 |
71 |
-- |
-- |
53 |
51 |
(56) |
56 |
61 |
(64) |
64 |
74 |
(66) |
62 ± 9 |
|
With S9 mix |
||||||||||
0.01 |
53 |
54 |
(54) |
NT |
NT |
-- |
NT |
NT |
-- |
54 a) |
0.05 |
59 |
41 |
(50) |
87 |
77 |
(82) |
81 |
76 |
(79) |
70 ± 17 |
0.1 |
46 |
59 |
(53) |
70 |
89 |
(80) |
72 |
62 |
(67) |
66 ± 14 |
0.2 |
NT |
NT |
83 |
87 |
(85) |
78 |
64 |
(71) |
78 ± 10 |
|
0.5 |
54 |
61 |
(58) |
87 |
91 |
(89) |
96 |
77 |
(87) |
78 ± 17 |
1 |
69 |
84 |
(77) |
106 |
100 |
(103) |
77 |
87 |
(82) |
87 ± 14** |
2 |
60 |
62 |
(61) |
69 |
97 |
(83) |
81 |
86 |
(84) |
76 ± 15 |
5 |
0 |
0 |
(0) |
31 |
40 |
(36) |
16 |
11 |
(14) |
16 ± 16 |
2AA 10 microg/plate |
272 |
358 |
(315) |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
2AA 25 microg/plate |
-- |
-- |
-- |
272 |
267 |
(270) |
276 |
286 |
(281) |
-- |
Negative control (air) |
49 |
63 |
-- |
57 |
66 |
-- |
52 |
59 |
-- |
-- |
56 |
60 |
(57) |
59 |
54 |
(59) |
56 |
54 |
(55) |
57 ± 4.7 |
|
With S9 mix and glutathione |
||||||||||
0.05 |
90 |
70 |
(80) |
128 |
98 |
(113) |
86 |
85 |
(86) |
93 ± 19** |
0.1 |
81 |
90 |
(86) |
91 |
96 |
(94) |
89 |
91 |
(90) |
90 ± 4.9** |
0.2 |
64 |
89 |
(77) |
123 |
107 |
(115) |
120 |
86 |
(103) |
98 ± 23** |
0.5 |
94 |
83 |
(89) |
131 |
146 |
(139) |
121 |
124 |
(123) |
117 ± 24** |
1 |
126 |
150 |
(138) |
108 |
109 |
(109) |
120 |
113 |
(117) |
121 ± 16** |
2 |
139 |
98 |
(119) |
100 |
129 |
(115) |
114 |
126 |
(120) |
118 ± 17** |
5 |
NT |
NT |
-- |
28 |
20 |
(24) |
17 |
24 |
(21) |
22 ± 4.8 |
2AA 10 microg/plate |
455 |
384 |
(420) |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
2AA 25 microg/plate |
-- |
-- |
-- |
153 |
141 |
(147) |
137 |
138 |
(138) |
-- |
Data shown are revertant colonies per plate. Induction of revertant colonies, ** p < 0.01 compared with results of negative control group in each strain. NT: not tested. AF2: 2 -(2 -furyl)-3 -(5 -nitro-2 -furyl)acrylamide, MMC: mitomycin C, 2AA: 2 -aminoantracene. a) Statistical analysis was not applied because number of measurements was less than three.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
A number of mutagenicity studies are available on the three constituents of Flux1. In this case one study has been selected for each constituent and each endpoint as the most representative and most relevant, and are summarised below. Overall, based on the data for the constituents, Flux1 is anticipated to provide negative results for mutagenicity and cytogenicity in somatic cells in vivo.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Adminstration of test material
1 or 2 times per gavage 24 hours apart
1 x by intraperitoneal route - Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: BDF1
- Sex:
- male
- Route of administration:
- other: 1 or 2 x per gavage, alternatively 1 x ip
- Duration of treatment / exposure:
- one or two gavage treatments 24 hours apart for bone marrow examination
single ip treatment for peripheral blood assay - Frequency of treatment:
- one or two gavage treatments 24 hours apart for bone marrow examination
single ip treatment for peripheral blood assay - Post exposure period:
- 24 hours after last treatment for bone marrow assessment
24, 48 and 72 hours after ip injection for peripheral blood assay - Remarks:
- Doses / Concentrations:
1000, 2000 and 3000 mg/kg intraperitoneal
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
500, 1000 and 2000 mg/kg by gavage
Basis:
nominal conc. - No. of animals per sex per dose:
- 5 per dose and treatment schedule
- Control animals:
- yes, concurrent vehicle
- Tissues and cell types examined:
- Bone marrow cell smears from femur, slides fixed with methanol and stained with 2.5 % Giemsa solution.
5 microL of blood placed on acridine orange-coated slide and covered. - Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information): negative
Carbon tetrachloride does not induce micronuclei and thus devoid of a mutagenic potential in this test system. - Executive summary:
Carbon tetrachloride was investigated in a mouse micronucleus test. Administrations were once or twice (24 hours apart) by gavage and sampling 24 hours later for the bone marrow assay or once by the ip route with samplings after 0, 24, 48 and 72 hours for the peripheral blood assay.
In none of the investigations were the micronucleated polychromatic erythrocytes (bone marrow) or micronucleated reticulocytes (peripheral blood) increased at any time point, while Mitmycin C showed the expected increases.
Carbon tetrachloride is devoid of a mutagenic potential in this test system.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Method development study with 20 other chemicals, low numbers of animals
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- GLP compliance:
- not specified
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Route of administration:
- oral: gavage
- Duration of treatment / exposure:
- single
- Frequency of treatment:
- once
- Post exposure period:
- 2 and 12 hours
- Remarks:
- Doses / Concentrations:
40, 400 mg/kg
Basis:
actual ingested - No. of animals per sex per dose:
- 3
- Control animals:
- yes, concurrent vehicle
- Tissues and cell types examined:
- hepatocytes
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information): negative
Carbon tetrachloride did not induce unscheduled DNA synthesis in this in vivo-in vitro system. - Executive summary:
Carbon Tetrachloride was investigated (together with 20 other chemicals) in the in vivo-in vitro UDS test. No UDS was detected at any of the two doses or time points.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Comparative non-standard test design and uncommon test strain, but following guideline procedure, based on scientific principles, sufficient documentation.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- , no positive control, an uncommon mouse strain though answering the Guideline criteria, and different sampling time.
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: Eµ-PIM-1 transgenic mice, lymphona prone
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Male and female (5 - 10 week old, 20 - 30 g) Eµ - PIM 1 transgenic mice were obtained from GenPharm International (Mountain View CA, U.S.A). Mice were randomized then housed in a clean air room with a 12 h light/dark cycle and given food ad libitum. Study animals had an acclimization period of 2-4 weeks.
- Route of administration:
- oral: gavage
- Vehicle:
- Corn oil (5 mL/kg bw)
- Details on exposure:
- - Treated and vehicle control animals were dosed daily (7 days per week) by oral gavage
- Corn oil suspensions of 1,2-dichloroethane were prepared weekly and were administered at 5 mL/kg bw
- Male and female mice were treated with 1,2-dichloroethane initially at 100 and 200 mg/kg bw (males) or 150 and 300 mg/kg bw (females) but the top doses were sequentially reduced by week 6, to 100 mg/kg bw for males and 150 mg/kg bw for females due to mortality and failure to gain weight. - Duration of treatment / exposure:
- 41 weeks (42 weeks were planned)
- Frequency of treatment:
- Once daily
- Post exposure period:
- No post exposure period
- Remarks:
- Doses / Concentrations:
100 and 200 mg/kg bw for males, 150 and 300 mg/kg bw for females. After the 6th week of treatment, 100 mg/kg bw for males and 150 mg/kg bw for females were administered.
Basis:
nominal conc. - No. of animals per sex per dose:
- 5 animals per sex, 10 animals (5 males and 5 females) per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No positive control was used
- Tissues and cell types examined:
- Peripheral blood; polychromatic (PCE) and normochromatic (NCE) erythrocytes
- Details of tissue and slide preparation:
- Peripheral blood (10-20 µL) was collected from a ventral tail vessel at 14 weeks and from the vena cava at terminal sacrifice. Blood smears were air dried then fixed for 10 min in absolute methanol. To reduce fading slides were scored within 1 week of staining with acridine orange. Coded slides were examined using a epifluorescence microscopy.
Slides from 10 animals per treatment group were analysed by one scorer for the number of micronucleated cells per 1000 NCE and the number of PCE per 1000 erythrocytes. - Evaluation criteria:
- Statistically significant compared to the control
- Statistics:
- Data from male and female mice were analysed separately using the Micronucleous Assay Data Management and Analysis System Software (Integrated Laboratory Systems, research triangle park, NC) as follows: The significance level was set at p ≤ 0.05. There was no evidence of significant interanimal variability in control groups so the number of MN-erythrocytes (MN-PCE or MN-NCE) were pooled across animals within each treatment group and assessed for dose effect using a one - tailed trend rest. a pairwise comparison between each treatment group and teh concurrent control was also done to determine the lowest effective dose. an ANOVA ratio was used to assess whether the percentages of PCE were significantly increased of reduced by treatment, indicating perturbation of erythropoiesis.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- , after 6 weeks of repeated dose of 200 mg/kg bw in males and 300 mg/kg bw in females per day, mortality and failure to gain weight were observed (therefore the dosage after week 6 was changed).
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not examined
- Additional information on results:
- Peripheral blood PCE frequencies were not affected by exposure to 1,2-dichloroethane at 14 weeks or at 41 weeks.
No treatment related increases in MN (micronucleus) - erythrocytes were seen in either sex at 14 weeks or at 41 weeks, when only NCE were analysed (because treatment had been stopped a week before sample collection). - Conclusions:
- Interpretation of results (migrated information): negative
There was no micronucleus induction or PCE suppression detected in the either sex after treatment with 100 to 300 mg/kg bw 1,2-dichloroethane in the presented study. Thus, under the conditions of the study, 1,2-dichloroethane was not considered to induce cytogenetic damage (chromosome damage). 1,2-dichloroethane therefore was not considered as a clastogenic substance. - Executive summary:
1,2 dichloroethane was examined for its possible cytogenetic effects (induction of chromosomal damage) using the micronucleus test, similarily conducted according to OECD Test Guideline 474 (Mammalian Erythrocytes Micronucleus Test) . Micronucleus (MN) induction in peripheral blood was examined in 10 male and female mice (lymphoma prone Eµ - PIM 1 transgenic mice) per dose after a repeated dose of 1,2 -dichloroethane in corn oil administered by gavage (5 mL/kg bw) once a day in concentrations of 100 and 200 mg/kg bw (in males) and 150 and 300 mg/kg bw (in females) up to week 6. After week 6 until the end of the exposure period (week 41) the top doses were reduced, therefore male animals were only administered with 100 mg/kg bw and females were only administered with 150 mg/kg bw (due to mortality and treatment related weight gain). Two negative controls were performed, a vehicle control and a “true” negative control with methyl cellulose. Peripheral blood was collected at week 14 and at terminal sacrifice (41 week) and analysed a week later (week 42). Stained slides (acridine orange staining) from 10 animals per treatment group were analysed for the number of micronucleated cells per 1000 NCE (normochromatic erythrocytes) and the number of PCE (polychromatic erythrocytes) per 1000 erythrocytes (Since exposure was discontinued one week before the terminal harvest of mice, only NCE were scored for micronuclei because of the short residence time of polychromatic erythrocytes.
Results showed no micronucleus induction (in normochromatic erythrocytes) after 14 weeks or 41 weeks of 1,2 -dichloroethane exposure and no polychromatic erythrocyte suppression was detected in the blood after 14 weeks or 41 weeks of exposure to the test substance. It can therefore be concluded that under the study conditions, 1,2 -dichloroethane induced no cytogenetic damage/chromosome damage.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September 2013 - May 2014
- Reliability:
- 1 (reliable without restriction)
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Draft OECD guideline: Rodent alkaline single cell gel electrophoresis (Comet) assay
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian comet assay
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- Species and Sex: Rat, female (nulliparous and nonpregnant)
Strain and Justification: F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, and the reliability of the commercial supplier.
Supplier and Location: Charles River (CR), (Kingston, New York)
Age at Study Start: approximately 10 weeks
Physical Acclimation: during the acclimation period, each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes
(fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two to three per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start of the study.
Housing: After assignment, animals were housed two-three per cage in stainless steel, solid bottom cages. Ground corn cob and shredded Aspen bedding was used in the cages. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
The following environmental conditions were maintained in the animal room:
Temperature: 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
Humidity: 40-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Enrichment: included the use of ground corn cob and shredded Aspen bedding and open areas on the cage sides for visualization of other rats. - Route of administration:
- inhalation: vapour
- Details on exposure:
- Groups of 28 female F344/DuCrl rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (six hours/day, seven days/week) for at least 28 exposures. Exposures were conducted under dynamic airflow conditions in Rochester-style stainless steel and glass whole-body inhalation exposure chambers. An additional group of three unexposed animals was administered MNU at 100 mg/kg, via oral gavage, approximately
three hours prior to the scheduled necropsy. This group of animals served as the positive control group for the Comet Assay. - Duration of treatment / exposure:
- Six hours/day, seven days/week for at least 28 exposures
- Remarks:
- Doses / Concentrations:
0 and 200 ppm
Basis:
nominal conc.
Rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (six hours/day, seven days/week) for at least 28 exposures. - No. of animals per sex per dose:
- Nine
- Control animals:
- yes
- Positive control(s):
- N-Nitroso-N-methylurea (MNU), 100 mg/kg bw
- Tissues and cell types examined:
- Mammary gland epithelial cells (MEC)
- Details of tissue and slide preparation:
- The Comet assay was conducted on MEC isolated from mammary tissue from nine rats/exposure group. Three additional rats were dosed with MNU at 100 mg/kg approximately three hours prior to the scheduled necropsy (these animals were not exposed in the inhalation chambers) to serve as the positive control for DNA damage. In addition to the positive control group, Trevigen CometAssay Control Cells for Alkaline Comet Assay (Trevigen
Inc., Gaithersburg, Maryland) were used in parallel with isolated MEC to serve as a technical control set for each Comet assay run. Cells from all preparations were processed for the Comet assay as recommended by the 4th International Comet Assay Workshop (Hartmann et al., 2003), and by following the outline in the Comet Assay Kit (Trevigen Inc., Gaithersburg, Maryland).
Epithelial Cell Isolation
This procedure was designed to effectively separate intact fat cells from mammary parenchymal tissue leaving viable epithelial cells to be analyzed using the Comet assay. Mammary tissue was excised and collected, excluding muscle and lymph nodes. The tissue was quickly weighed and placed in an appropriate amount of ice-cold Hank’s Balanced Salt Solution (HBSS calcium/magnesium free (CMF), Gibco, Grand Island, New York) to thoroughly wash the tissue and to remove any residual blood or fur. After washing twice, all pieces of tissue were minced using scissors for approximately 5 minutes (tissue had a slightly paste-like consistency) on a watch glass placed on foil over a bed of ice. A protease cocktail solution (Rat Breast Tissue Dissociation System, Breast OptiTDS, CHI Scientific, Maynard, Massachusetts) containing collagenase type I, collagenase type III, collagenase type IV from Clostridium histolyticum, and trypsin from bovine pancreas was prepared fresh and used to digest the mammary tissue. The fresh enzyme working solution was made by combining 1.0 ml of the Breast Tissue Dissociation System, OptiTDS enzyme mix with 9 ml of the Breast OptiTDS Digestion Buffer (maximum of 4-5 g of tissue per 10 ml diluted enzyme working solution). This diluted working enzyme solution was warmed at approximately 37°C for approximately 10 minutes prior to use. The fully minced tissue was placed in the pre-warmed enzyme solution and digested in a loosely capped 50 ml glass bottle in a shaking incubator (150 rpm) at approximately 37°C for approximately 30 minutes. At the end of the digestion period, the tissue digest was gently triturated with a 10 ml pipette 5-6 times to further dissociate the tissue (pipette was rinsed first with 10% BSA to prevent cells from sticking
to the pipette). The digest was first passed through one layer of a standard gauze pad and then carefully passed through nylon mesh (100 μm) using a pipette and brought up to a final volume of 30 ml with ice-cold CMF-HBSS. This homogenate was centrifuged at 201 x g for approximately 5 minutes at approximately 4°C with the brake on. The supernatant, containing mostly adipose tissue, was carefully aspirated off and discarded. The pellet (epithelial cells and fibroblasts) was resuspended in ice-cold 2% fetal bovine serum (FBS) in approximately 20 ml of CMF-HBSS then centrifuged at 80 x g for approximately 1 minute at approximately 4°C with the brake off (supernatant = fibroblasts; pellet = epithelial cells). The supernatant was carefully removed and the pellet resuspended in 30 ml of ice-cold CMF-HBSS and then centrifuged at 201 x g for approximately 5 minutes at approximately 4°C with the brake on. Again, the supernatant was removed, and the pellet resuspended in 5 ml of ice-cold CMF-HBSS. This final suspension was used to verify cell number and viability (Trypan blue exclusion method) and to ultimately perform the Single Cell Gel Electrophoresis assay (Comet assay).
Slide Preparation
From the above prepared MEC isolation suspensions, an aliquot of 50-75 μL was mixed with low melting agarose (0.5%). The cell/agarose suspension was applied to a Trevigen Comet assay glass microscope slide containing two spots with normal melting agarose (1%). These slides were cooled at room temperature for 2-10 minutes then placed at approximately 2-8°C for 20-30 minutes to solidify. Two slides were prepared for each treatment with one slide serving as a backup.
Lysis
The slides were submerged in pre-chilled lysis solution and remained in this solution overnight at approximately 2-8°C.
Unwinding
After the lysis treatment, slides were carefully rinsed in deionized water (diH2O) to remove residual detergents and then transferred into freshly prepared, refrigerated (approximately 2-8°C) alkaline solution, pH>13, for approximately 20 minutes in the dark. This allowed the DNA to unwind. A constant amount of time for unwinding was allotted throughout the experiment.
Electrophoresis
Using the same alkaline buffer and under the same conditions (approximately 2-8°C, protected from light), electrophoresis was conducted for approximately 20 minutes at 0.7 volts/cm. Electrophoresis was conducted using the CometAssay Electrophoresis System (Trevigen).
Wash
After electrophoresis, the slides were rinsed by dipping several times in diH2O and then dehydrated by immersion in 70% ethanol for approximately 5 minutes. The slides were air dried and stored at room temperature until analyzed.
Staining
Slides that were designated for scoring were stained with a DNA stain (SYBR Gold, Life Technologies, Carlsbad, California) prior to scoring. The stain solution was prepared by diluting 7.5 μL of SYBR Gold stain in 15 ml of 1xTE (tris EDTA buffer solution). - Evaluation criteria:
- Scoring (Comet visualization and analysis)
Each slide was uniquely identified with a random code prior to being scored. Fifty cells (comets) per well were scored, resulting in a total of 100 cells evaluated per replicate (conducted in triplicate). If the primary slide did not have 50 scorable cells per well or 100 total cells, additional cells were scored using the backup slide. The comets were viewed on a Leica/Leitz DMRX fluorescence microscope at 200X magnification with a digital camera linked to an image analysis system (Comet IV v 4.11 from Perceptive Instruments Ltd., UK). The % tail DNA (also known as % tail intensity or % DNA in tail), defined as the percentage of DNA fragments present in the tail relative to the percentage of DNA fragments in the head, was the
parameter measured in this study. Each slide was examined for possible indications of cytotoxicity, i.e., presence of ‘clouds.’ The rough estimate of the percentage of clouds was recorded for each slide, but not included in the calculation for DNA damage. The clouds, also known as “hedgehogs,” are morphologically indicative of highly damaged cells often associated with severe genotoxicity, necrosis, or apoptosis, and were not scored. Morphologically, clouds consist of a small or nonexistent head and a large diffuse tail (Collins, 2004). For purposes of this study, any comet having a “% tail DNA” value of >95 was classified as a cloud. - Sex:
- female
- Genotoxicity:
- negative
- Remarks:
- No exposure-related genotoxic effects in the Comet assay with mammary gland epithelial celles were observed.
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No exposure-related genotoxic effects in the Comet assay were observed.
- Conclusions:
- Interpretation of results (migrated information): negative No exposure-related genotoxic effects in the Comet assay were observed.
The lack of any exposure-related genotoxic effects in the Comet assay or relevant target-tissue specific DNA adducts does not support a genotoxic/mutagenic Mode of Action for DCE-induced mammary tumors in rats. . - Executive summary:
The purpose of this study was to investigate the potential mode of action (MoA) of 1,2-dichloroethane (DCE)-induced mammary tumors in female F344/DuCrl rats. Groups of 28 female F344/DuCrl rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (six hours/day, seven days/week) for at least 28 exposures. Exposures occurred under dynamic airflow conditions in whole-body inhalation exposure chambers. An additional group of three animals were administered N-Nitroso-N-methylurea (MNU) at 100 mg/kg, via oral gavage, approximately three hours prior to the scheduled necropsy. This group of animals served as the positive control group for the Comet Assay. An additional group of six animals were administered 750 mg/kg diethyl maleate (DEM) via intraperitoneal (i.p.) injection approximately two hours prior to necropsy to serve as a positive control for depletion of glutathione (GSH) in mammary and liver tissue. Study parameters measured included cage-side and clinical observations, feed consumption, body weights/body weight gains, estrous evaluations, serum prolactin levels, measurement of reduced (GSH) and oxidized (GSSG) glutathione, DCE-glutathione conjugates S-(2-Hydroxyethyl)glutathione hydrochloride (HESG) and S,S’-Ethylene-bis- glutathione (EBG), DNA adducts 8-Hydroxy-2’-deoxyguanosine (8-OH dG) and S-(2-guanylethyl) glutathione (GEG) in mammary and liver tissue, Comet assay (mammary tissue), morphometric evaluation of mammary gland structure, cell proliferation (Ki-67), and histopathology (mammary tissue).
All rats were sacrificed immediately after exposure on the first diestrus after a minimum of 28 consecutive days of exposure. This resulted in a range of 28-31 days of exposure to average chamber concentrations of 0.0 ± 0.0 (control) or 205.1 ± 4.2 ppm DCE (study mean ± standard deviation). The range in exposure days had no measurable impact on any study parameter. Animals exposed to 200 ppm DCE had treatment-related decreases in body weight gain from test day (TD) 1-15 with correlating decreases in feed consumption from TD 1-8; however, body weight gain and feed consumption were similar to control for the remainder of the study.
Under the conditions of this study, repeated inhalation exposure to a high concentration of DCE vapors had no effect on body weights, clinical observations, serum prolactin levels, mammary epithelial cell proliferation/numeric density, or mammary gland morphology or histopathology. Based on the results of the Comet assay, no exposure related DNA damage was detected in mammary epithelial cells (MEC) isolated from DCE-exposed rats, compared to control rats. Compared to control rats, repeated inhalation of DCE had no effect on GSH or GSSG levels in mammary tissue; however, DCE exposure decreased liver (non-target tissue) GSH and GSSG levels by approximately 72 and 62%, respectively. The GSH/GSSG ratio remained essentially unchanged.
No HESG or EBG was measured at levels greater than the lower limit of quantitation (LLQ) in mammary or liver tissue samples (LLQ= 10 and 10 ng/g tissue for both mammary and liver tissue) isolated from control or DCE-exposed rats. Compared to control rats, DCE exposure had no effect on 8-OHdG adduct levels in mammary tissue; however, liver 8-OHdG levels in DCE-exposed rats were significantly less than control rats.
In contrast to the 8-OHdG adduct, endogenous GEG adduct was not quantifiable in mammary or liver tissue isolated from control rats, with a lower limit of quantitation of 0.6 ng/mL adducts/106 dG for both mammary and liver tissue. Compared to control rats a statistically significant DCE-dependent increase in GEG levels in both mammary (~2.4-fold; 103 ± 16 GEG adducts/106 dG residues) and liver (4.3-fold; 222 ± 41 GEG adducts/106 dG residues) was observed. Thus the GEG adduct levels in the non-target tissue (liver) were approximately ~54% higher than in the mammary (target) tissue from the same DCE-exposed rats.
In conclusion, repeated inhalation exposure to 200 ppm DCE vapor, a concentration approximately 20% higher than the concentration reported to induce mammary tumors in rats (Nagano et al., 2006) had no statistically significant effect on serum prolactin levels, GSH/GSSG levels, cell proliferation, or DNA damage in mammary tissue. The N7-guanylethyl glutathione (GEG) cross-link adduct was identified as a biomarker of exposure, with higher levels of the GEG adduct measured in (non-target) liver tissue compared to (target) mammary tissue isolated from the same rats. The results of this sub-acute inhalation exposure study do not support a specific known MoA for DCE-induced mammary tumors in rats. However, the lack of any exposure-related genotoxic effects in the Comet assay or relevant target-tissue specific DNA adducts does not support a genotoxic/mutagenic MoA.
- Endpoint:
- in vivo mammalian germ cell study: gene mutation
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1999
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Well-conducted study, but no guideline and GLP.
- Qualifier:
- no guideline available
- GLP compliance:
- not specified
- Type of assay:
- transgenic rodent mutagenicity assay
- Species:
- mouse
- Strain:
- other: MutaMice (TM)
- Sex:
- male
- Route of administration:
- other: oral gavage or intraperitoneal
- Vehicle:
- Olive oil
- Details on exposure:
- The animals were given a single injection, intraperitoneally (ip) or by oral gavage (po): 75 or 150 mg DCE per kg.
In consecutive administration experiments, intraperitoneal injection was carried out once a day for 5 days a week. DCE was injected at 40 mg/kg/injection five times (200 mg/kg in total) or at 20 mg/kg/injection for first six injections and at 40 mg/kg/injection for the following four days (280 mg/ kg in total). - No. of animals per sex per dose:
- 3
- Control animals:
- yes, concurrent vehicle
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Negative controls validity:
- valid
- Positive controls validity:
- not valid
- Conclusions:
- Interpretation of results (migrated information): negative
No increase was detected in the frequency of lacZ mutations in the liver and testis of Muta™Mouse following 1,2-dichloroethane administration of single doses of up to 150 mg/kg or of consecutive injections of up to 280 mg/ kg. - Executive summary:
Possible induction of lacZ mutation was examined in the liver and testis of Muta™Mouse following the administration of carcinogenic halogenated compounds, namely 1,2-dichloroethane (DCE), 1,2-dibromoethane (DBE), carbon tetrachloride, or 1,2-dibromo-3-chloropropane (DBCP). Slight increases were observed on the mutant frequency in the testis DNA isolated from the mice 14 days after treatment with DBCP at 40 mg/kg or with DBE at 60 mg/kg but not in the liver. Further investigation was necessary to confirm the mutation induction by these chemicals in the testis including experiments with longer sampling intervals. No increase was detected in the frequency following DCE administration of single doses of up to 150 mg/kg or of consecutive injections of up to 280 mg/ kg. Marginal but biologically insignificant responses were observed in the liver from the carbon tetrachloride exposed mice. The present results suggest that these carcinogenic chemicals are less efficient for induction of gene mutation in the liver of Muta™Mouse.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: The study was carried out under GLP conditions and in accordance with OECD Guideline for Testing of Chemicals No. 474 (1997) without restrictions.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, UK
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 183 to 316 g for males; 172 to 225 g for females
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: up to six of the same sex in cages
- Diet (e.g. ad libitum): access ad libitum to SQC Rat and Mouse Maintenance Diet No. 1, Expanded (Special Diets Services Ltd., Witham)
- Water (e.g. ad libitum): Mains water provided ad libitum
- Acclimation period: 5 to 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness - Route of administration:
- oral: gavage
- Vehicle:
- The test article was dissolved in corn oil
- Details on exposure:
- The test article and vehicle were weighed into suitable containers and added to a designated dose pot and stirred until mixed to make the final volume. A two hour expiry was added to each formulation following dissolution. The dosing preparations had concentrations of 12, 24 and 48 mg/mL. A total of 10 mL of each dosing preparation was administered by oral gavage to test animals.
- Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- Once daily
- Post exposure period:
- 24 hours
- Remarks:
- Doses / Concentrations:
120 mg/kg body weight
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
240 mg/kg body weight
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
480 mg/kg body weight
Basis:
nominal conc. - No. of animals per sex per dose:
- 6 males, 6 females
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- The clastogenic positive control was cyclophosphamide (CPA, Sigma Chemical Co, Poole, UK) freshly prepared in saline. The aneugenic positive control was carbendazim (CBZ Sigma Chemical Co, Poole, UK) freshly prepared in 1 % (w/v) aqueous methylcellulose (1 % MC).
- Tissues and cell types examined:
- Body weights were recorded on each day of dosing and on the post-dose observation day. Body temperatures were recorded approximately 2 hours following the doses on Day 1 and Day 5. Blood samples were taken from satellite animals dosed by the same route, dose levels and at the same dosing frequency as that described for the micronucleus animals.
- Details of tissue and slide preparation:
- Test article, vehicle and CBZ-treated rats were sampled in groups, 24 hours after the final administration; CPA treated rats were sampled 24 hours after a single dose. Rats were killed by an overdose of sodium pentobarbitone, given via intraperitoneal injection and subsequently ensured by cervical dislocation, in the same sequence used for dosing.
One femur (Range Finder Experiment) or both femurs (Micronucleus Experiment) were exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes.
The bone marrow suspensions from the Range Finder animals and one set of suspensions (from one femur per animal) from the Micronucleus Experiment were processed and slides prepared as follows:
A further 3 mL of foetal bovine serum was added to the tubes, which were then centrifuged at 200 g for approximately five minutes; the serum was aspirated to leave one or two drops and the cell pellet. It should be noted that a second washing step was performed (resuspending the cell pellet in 3 mL of serum), where considered necessary to optimise slide quality.
The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. The latter served as the code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were allowed to air dry and then fixed for 10 minutes in absolute methanol. Slides were allowed to dry (and stored at room temperature until required for staining). Staining was performed on the same day as slide preparation. One slide from each set was taken (any remaining slides were kept in reserve). Prior to staining any stored slides were fixed again for 10 minutes in absolute methanol. After rinsing several times in distilled water, slides were stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis.
Additional spare slides from female animals were stained and analysed from animal numbers 990 (vehicle), 955 (120 mg/kg/day), 966 (240 mg/kg/day), 970, 976, 967 (CPA), and 981 (Carbendazim, 1500 mg/kg/day). This was due to sub-optimal staining of initial slides.
In order to allow optimum slide preparation for potential antikinetochore (AK) staining, bone marrow collected from the second femur per animal was passed through a cellulose filtration system to remove 60%-70% of the nucleated cell fraction. This procedure is outlined below:
Bone marrow was sampled as per previously described into 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. An additional 4 mL of serum was added to each bone marrow sample prior to adding to pre-prepared cellulose filtration columns.
The cellulose columns contained 2 mL of an equal mix of type 50 and cellulose achieving a 50 mg/mL solution. These were prepared the day prior to filtration. Preparation of the columns was in accordance to SOP MT 45/030 and as recommended by published data [ ].
Once filtered the bone marrow cells were centrifuged at 200 x ‘g’ for 5 minutes at room temperature and standard smears prepared. Two to three slides were prepared per animal in this way depending on the quantity of bone marrow available.
Following fixation, prepared slides were stored in slide boxes at nominal 20C prior to possible further AK staining.
Due to the negative micronucleus data obtained from this study, further mechanistic analysis was not required. As such, filtered slides were not processed further. - Evaluation criteria:
- The assay was considered valid if all the following criteria were met:
1. The incidence and distribution of MN PCE in vehicle control groups were consistent with the laboratory’s historical vehicle control data, and
2. The proportion of immature erythrocytes among total erythrocytes (expressed as %PCE) should not be less than 20% of the control value at each test article dose, and
3. At least five animals (per sex) out of each group (males and females) were available for analysis, and
4. The positive control chemical (CPA) induced a statistically significant increase in the frequency of MN PCE.
Acceptance under any other criteria are discussed in the results section.
For valid data, the test article was considered to induce micronuclei if:
1. A statistically significant increase in the frequency of MN PCE occurred at one or more dose levels
2. The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
3. A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were to be dealt with on a case by case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result [ ]. Biological relevance was taken into account, for example consistency of response within and between dose levels. - Statistics:
- After completion of microscopic analysis and decoding of the data the following were calculated:
1. % PCE for each animal and the mean for each group. The group mean % PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per 2000 PCE) and % MN PCE for each animal and the group mean % MN PCE (+/- standard deviation).
The numbers of MN PCE in vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable. For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square test.
The numbers of micronucleated PCE in each treated group (males and females separately) were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p < 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
As the heterogeneity chi-square test provided evidence of significant (p < 0.05) variability between animals within at least one group, non-parametric analysis by use of the Wilcoxon rank sum test was performed. This was conducted on male and female data. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 240 and 480 mg/kg body weight
- Clinical signs of toxicity in test animals: Clinical signs of toxicity in the 240 mg/kg dose group included decreased activity, piloerection, ptosis, mouth rubbing, paddling and hunched posture and similar effects plus eye-closure, ataxia and unkempt appearance at 480 mg/kg, which persisted longer than in the lower dose group.
- Evidence of cytotoxicity in tissue analyzed: Rats treated with chloroform showed group mean PCE/NCE ratios (expressed as % PCE) that decreased in a dose dependent manner, with the highest dose exhibiting 38 % PCE (males) or 27 % PCE (females). These ratios were markedly lower than the concurrent vehicle control values of 62 % or 44 % PCE (males and females respectively). These data were also lower than historical values and as such were considered to represent some evidence of bone marrow toxicity and target organ exposure.
- Rationale for exposure: Data from a bone marrow chromosome aberration study indicated statistically significant increases in structural chromosome aberrations following five days of oral exposure
- Harvest times: The duration of the study has been shown to be of sufficient duration for the expression of any genotoxic potential.
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE
- Ratio of PCE/NCE (for Micronucleus assay): The polychromatic erythrocytes to normal erythrocytes ratio of negative (vehicle) control male rats was slightly higher than that of the historical vehicle control (normal) range (62 % PCE versus 39 to 59 %). The ratio was within normal values for control females. As individual frequencies of micronucleated PCE were consistent with historical vehicle control distribution data (both genders) and vehicle treated animals did not exhibit clinical signs of toxicity, the vehicle data were accepted as valid. The clastogen cyclophosphamide positive control group exhibited significantly increased numbers of micronuleated PCE, which was also seen in the aneugenic Carbendazim positive control group.
- Appropriateness of dose levels and route: The results of the bioanalysis confirm that animals dosed at 120, 240 and 480 mg/kg body weight were systemically exposed to chloroform
- Statistical evaluation:
1. The incidence and distribution of MN PCE in the vehicle control group were consistent with the laboratory's historical vehicle control data. This was apparent for female animals but not for males where the group mean %PCE slightly exceeded historical values (62% PCE versus 39%-59% range). The reason for this high value was not clear but may have been due to staining characteristics. As this increase was small, with individual frequencies of MN PCE consistent with historical vehicle control distribution data, with animals showing no clinical observations of ill health, these data were accepted as valid.
2. The proportion of immature erythrocytes among total erythrocytes (expressed as %PCE) were not less than 20% of the control value at each test article dose analysed.
3. At least five animals out of each group (male and female) were available for analysis.
4. The positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE (Appendix 6).
The assay data were therefore considered valid. - Conclusions:
- Interpretation of results (migrated information): negative
Chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female rats treated up to 480 mg/kg body weight/day for five consecutive days, under the experimental conditions employed. - Executive summary:
Chloroform was tested for its ability to induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female young adult Sprague Dawley rats, following 5 days of repeat oral dosing. The test was carried out under GLP conditions and in accordance with OECD Guideline No. 474. From range finding tests, 480 mg/kg body weight/day was considered a suitable maximum tolerated dose under the assay conditions. The final micronucleus study was performed by administration by oral gavage of 120, 240 or 480 mg/kg body weight/day chloroform in corn oil for five consecutive days to groups of six male and six female rats, respectively. Analyses of formulations administered to animals demonstrated variability in terms of achieved concentrations from all of the sampling points across the range of concentrations used and most particularly at the low dose level (-9.9 to -91.4 % of the nominal concentration of 12 mg/mL; -4.3 to -36.4 % of nominal 24 mg/mL; -2.7 to -20.3 % of nominal 48 mg/mL used for dosing). However, the analyses of blood plasma confirmed that animals were systemically exposed to chloroform with increasing exposure with both concentration and time. Two additional groups of six males and six females were treated once with a clastogen positive control, cyclophosphamide, 24 hours prior to necropsy (20 mg/kg). Two groups of six males and six females were treated twice with an aneugenic positive control, Carbendazim (1500 mg/kg and 2000 mg/kg). During the treatment period, clinical signs observed essentially in the 480 mg/kg/day group included among others ataxia, hunched posture, hypothermia, lethargy, decreased activity, ptosis, piloerection and tremors. The group mean frequency of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE), ratio expressed as % PCE, of the negative vehicle controls were considered as valid, although negative male control rats exhibited a slightly increased ratio. The positive control groups exhibited significantly increased frequencies of micronucleated PCE in comparison with the concurrent controls. The assay system was therefore considered as valid. Rats treated with chloroform showed group mean % PCE values that decreased in a dose dependent manner, with the highest dose group (480 mg/kg/day) exhibiting 38 % PCE (males) or 27 % PCE (females). These were markedly lower than the concurrent vehicle control values of 62 % or 44 % PCE (male and females respectively) and also lower than historical values and as such were considered to represent some evidence of bone marrow toxicity and target organ exposure. The group mean frequencies of micronucleated PCE observed in the groups treated with chloroform were not significantly different to the vehicle controls. In addition, individual frequencies of micronucleated PCE were generally similar to those seen in the vehicle control groups and consistent with the laboratory's historical control distribution data. From the results of the study it is concluded that chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female Sprague Dawley rats treated up to 480 mg/kg body weight/day for five consecutive days, under the experimental conditions employed. Thus, chloroform was not genotoxic in the present micronucleus assay.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- GLP compliance:
- no
- Type of assay:
- unscheduled DNA synthesis
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Labs (Kingston, NY)
- Age at study initiation: no data
- Weight at study initiation: (175-275 g
- Diet (e.g. ad libitum): NIH-07 feed (Ziegler Brothers, Gardner, PA) - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- Rats were treated by gavage with chloroform in corn oil. The total volume of the test solution administered was 0.1-0.4 ml/ 100 gm body weight. The highest dose is generally selected to be at or near the LD50.
- Duration of treatment / exposure:
- single treatment
- Frequency of treatment:
- single treatment
- Post exposure period:
- 48 hours
- Remarks:
- Doses / Concentrations:
0, 40, 400 mg/kg body weight
Basis:
nominal conc. - No. of animals per sex per dose:
- three males per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Among others Aflatoxin B1, 2-acetylaminofluorene (2-AAF), dimethylnitrosamine (DMN), N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- Tissues and cell types examined:
- Viable male rat hepatocytes
- Details of tissue and slide preparation:
- Livers were perfused in situ for 2.5 min at 20 mL/min with a 0.5-mM solution of ethyleneglycolbis N, N'tetraacetic acid in Hanks balanced salt solution without Ca+2 or Mg+2 followed by 12 min at 20 mL/min of a 100 units/mL solution of Type 1 collagenases in Williams medium E. A single-cell suspension of hepatocytes was obtained by combing out cells in a petri dish of collagenase solution. Cells were collected by a 5-min centrifugation at 50 g, resuspended in ice cold WE, and filtered through 4-ply sterile gauze to remove debris. Approximately 6 x 10^5 viable cells were seeded into Linbro 35-mm 6 well cluster dishes containing 25 mm round plastic coverslips and 4 mL WE containing 10 % fetal calf serum. All perfusion and culture solutions were supplemented with 50 ug/mL gentamicin. The total elapsed time from initiation of the perfusion to the time cells were placed in media did not exceed 60 min. Cells were incubated for 90 +/- 10 min at 37 °C in a 95 % air-5% CO2 incubator to allow attachment of cells to the coverslips. Cultures were washed once with WE and incubated in 2 mL WE containing 10 uCi/mL 3H-thymidine buffered with HEPES (final concentration 0.01 M) for 4 hr. Cultures were washed once with WE and incubated overnight (14-16 hr) in 0.25 mM thymidine in WE buffered to a final concentration of 0.01 M HEPES.
Cultures were washed twice with WE followed by 10 min in 1 % sodium citrate, 3 x 10 min in 1:3 glacial acetic acid/absolute ethanol, and six washes with deionised, distilled water. When dry, coverslips were mounted to microscope slides and dipped in Kodak NTB-2 emulsion diluted 1:1 with water. Slides were exposed for 12-14 days at -20 °C then developed for 3 min in Kodak D-19 developer at 15 °C, u min in Kodak fixer, and washed in water for 25 min. Cells were stained for 20-30 sec in 1 % solution methyl-green Pyronin Y. A coverslip was mounted over the cells with Permount. - Evaluation criteria:
- A colony counter was interfaced to a microscope via video camera. An area of the slide was randomly selected and 50 morphologically unaltered cells were counted. The highest three nuclear-sized areas over the cytoplasm adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). The cells in repair are those exhibiting >= 5 NG.
- Statistics:
- no data
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Remarks:
- the highest dose was generally selected to be at or near the LD50
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Cells from negative control animals (given vehicle only) ranged from -3.0 to -5.1 net grains/nucleus. CHCl3 at 40 and 400 mg/kg yielded a negative response (-2.7 to -4.4 net grains/nucleus). All the genotoxic hepatocarcinogens tested (dimethylnitrosamine, 2-acetylaminofluorene...) produced strong positive response of >15.
- Conclusions:
- Interpretation of results (migrated information): negative
Oral exposure to chloroform by gavage at doses of 40 or 400 mg/kg bodyweight did not cause genotoxicity in male Fischer-344 rats up to 12 hours after a single dose as revealed by the in vivo-in vitro hepatocyte DNA repair assay. - Executive summary:
The present study investigated the genotoxicity of chloroform in male Fischer-344 rats exposed orally via gavage to 0, 40, or 400 mg/kg body weight. Genotoxicity was tested by the measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS).
Rats were treated by gavage with chloroform in corn oil, and the total volume of the test solution administered was 0.1 -0.4 mL/100 gm bodyweight. UDS was measured by counting grains by quantitative autoradiography. The highest of three nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to give the net grains/nucleus (NG). The percentage of cells in repair is defined as those exhibiting => 5 NG.
Cells from negative control animals (given corn oil only) ranged from -3.0 to -5.1 net grains/nucleus. CHCl3 at 40 and 400 mg/kg yielded a negative response (-2.7 to -4.4 net grains/nucleus).
Referenceopen allclose all
Bone marrow test (Table 1):
There was no increase in the frequency of micronucleated polychromatic erythrocytes due to the treatment of carbon tetrachloride after either one or two treatments. Mitomycin C (single treatment) produced a statistically significant increase at all three time points .
Table 1: Bone marrow micronucleus test of carbon tetrachloride with oral gavage:
| Dose (mg/kg) | No of mice | No of injections | mean Micronuc. PCEs | PCE ratios | |
| 0 (corn oil) | 5 | 1 | 0.20 +/- 0.16 | 38.1 +/- 5.3 | |
| 500 | 5 | 1 | 0.20 +/- 0.10 | 27.4 +/- 6.2 | |
| 1000 | 5 | 1 | 0.38 +/- 0.18 | 29.7 +/- 10.7 | |
| 2000 | 5 | 1 | 0.22 +/- 0.16 | 24.8 +/- 5.0 | |
| Mitomycin C 0.5 mg/kg | 5 | 1 | 1.58 +/- 0.61 (p< 0.01) | 39.7 +/- 3.2 | |
| 0 (corn oil) | 5 | 2 | 0.12 +/- 0.04 | 36.0 +/- 3.5 | |
| 500 | 5 | 2 | 0.28 +/- 0.16 | 26.0 +/- 3.9 | |
| 1000 | 5 | 2 | 0.28 +/- 0.22 | 22.5 +/- 9.6 | |
| 2000 | 5 | 2 | 0.20 +/- 0.19 | 20.8 +/- 8.6 |
Peripheral blood test (Table 2):
There was no inclrease in the frequency of micronucleated reticulcytes due to the teatment of carbon tetrachloride at any observation time point. Mitomycin C produced a statistically significant increase
Table 2: Peripheral blood micronucleus test of carbon tetrachloride with intraperitoneal injection
| Dose (mg/kg) | No of mice | Treatment time | % Micronucl. RET, means +/- SD |
| 1000 | 5 | 0 | 0.20 +/- 0.07 |
| 5 | 24 | 0.24 +/- 0.09 | |
| 5 | 48 | 0.20 +/- 0.12 | |
| 5 | 72 | 0.26 +/- 0.17 | |
| 2000 | 5 | 0 | 0.18 +/- 0.13 |
| 5 | 24 | 0.18 +/- 0.16 | |
| 5 | 48 | 0.26 +/- 0.11 | |
| 5 | 72 | 0.28 +/- 0.13 | |
| 3000 | 5 | 0 | 0.16 +/- 0.09 |
| 5 | 24 | 0.26 +/- 0.05 | |
| 5 | 48 | 0.20 +/- 0.10 | |
| 5 | 72 | 0.20 +/- 0.16 | |
| Mitomycin C | 5 | 0 | 0.16 +/- 0.09 |
| 1 mg/kg | 5 | 24 | 0.56 +/- 0.34 (p<0.01) |
| 5 | 48 | 3.90 +/- 0.67 (p<0.01) | |
| 5 | 72 | 0.96 +/- 0.37 (p<0.01) |
Carbon tetrachloride did not induce unschduled DNA synthesis in this test system
Table 1: Induction of UDS by Carbon tetrachloride in the In Vivo-In Vitro Hepatocyte DNA Repar Assay
| Chemical | Dose (mg/kg) | Time (hr) | n | nuc. grains +/- SE | % IR +/- SE |
| CCl4 in corn oil | 40 | 2 | 3 | -3.1 +/- 0.3 | 5 +/-4 |
| 400 | 2 | 3 | -3.6 +/- 0.6 | 4 +/-4 | |
| 400 | 12 | 3 | -4.0 +/- 1.2 | 4 +/-2 | |
Corn oil |
2 | 7 | -5.1 +/- 0.5 | 1 +/- 0 | |
| 12 | 13 | -4.4 +/- 0.5 | 3 +/-1 |
CCl4 induced DNA replication, at the dose of 400 mg/kg 4.1 % of the cells were in S-phase, whereas the control value was 0.08 .
No remarks
Body weights in all groups and body weight gains of MNU-treated animals (Comet assay positive control group) were similar to controls throughout the duration of the study. There were no treatment-related clinical observations noted on any other animals throughout the duration of the study.
Table 1: Individual animal micronucleus frequencies - males
Treatment (mg/kg/day) |
Animal Number |
PCE Count |
NCE Count |
% PCE |
Total PCE Count |
MN PCE |
% MN PCE |
Vehicle |
469 |
638 |
362 |
63.80 |
2000 |
5 |
0.25 |
467 |
525 |
475 |
52.50 |
2000 |
1 |
0.05 |
|
456 |
736 |
264 |
73.60 |
2000 |
0 |
0.00 |
|
473 |
482 |
518 |
48.20 |
2000 |
0 |
0.00 |
|
452 |
642 |
358 |
64.20 |
2000 |
1 |
0.05 |
|
465 |
721 |
279 |
72.10 |
2000 |
1 |
0.05 |
|
120 |
458 |
657 |
343 |
65.70 |
2000 |
3 |
0.15 |
450 |
846 |
154 |
84.60 |
2000 |
3 |
0.15 |
|
468 |
610 |
390 |
61.00 |
2000 |
1 |
0.05 |
|
471 |
706 |
294 |
70.60 |
2000 |
3 |
0.15 |
|
447 |
623 |
377 |
62.30 |
2000 |
2 |
0.10 |
|
437 |
425 |
575 |
42.50 |
2000 |
1 |
0.05 |
|
240 |
454 |
401 |
599 |
40.10 |
2000 |
1 |
0.05 |
451 |
493 |
507 |
49.30 |
2000 |
1 |
0.05 |
|
475 |
590 |
410 |
59.00 |
2000 |
2 |
0.10 |
|
463 |
433 |
567 |
43.30 |
2000 |
0 |
0.00 |
|
476 |
571 |
429 |
57.10 |
2000 |
1 |
0.05 |
|
445 |
458 |
542 |
45.80 |
2000 |
1 |
0.05 |
|
480 |
464 |
522 |
478 |
52.20 |
2000 |
1 |
0.05 |
462 |
405 |
595 |
40.50 |
2000 |
1 |
0.05 |
|
474 |
164 |
836 |
16.40 |
2000 |
4 |
0.20 |
|
470 |
Data Omitted* |
||||||
466 |
334 |
666 |
33.40 |
2000 |
0 |
0.00 |
|
442 |
486 |
514 |
48.60 |
2000 |
4 |
0.20 |
|
CPA, 20+ |
472 |
461 |
539 |
46.10 |
2000 |
34 |
1.70 |
446 |
620 |
380 |
62.00 |
2000 |
53 |
2.65 |
|
461 |
408 |
592 |
40.80 |
2000 |
23 |
1.15 |
|
457 |
401 |
599 |
40.10 |
2000 |
41 |
2.05 |
|
443 |
490 |
510 |
49.00 |
2000 |
47 |
2.35 |
|
459 |
490 |
510 |
49.00 |
2000 |
29 |
1.45 |
|
+ administered as a single dose; * animal killed in extremis, data omitted; MN micronucleated
Table 2: Individual animal micronucleus frequencies - females
Treatment (mg/kg/day) |
Animal Number |
PCE Count |
NCE Count |
% PCE |
Total PCE Count |
MN PCE |
% MN PCE |
Vehicle |
968 |
397 |
603 |
39.70 |
2000 |
0 |
0.00 |
992 |
427 |
573 |
42.70 |
2000 |
4 |
0.20 |
|
995 |
470 |
530 |
47.00 |
2000 |
3 |
0.15 |
|
990 |
443 |
557 |
44.30 |
2000 |
2 |
0.10 |
|
979 |
410 |
590 |
41.00 |
2000 |
4 |
0.20 |
|
959 |
482 |
518 |
48.20 |
2000 |
1 |
0.05 |
|
120 |
973 |
417 |
583 |
41.70 |
2000 |
2 |
0.10 |
963 |
445 |
555 |
44.50 |
2000 |
2 |
0.10 |
|
955 |
421 |
579 |
42.10 |
2000 |
2 |
0.10 |
|
977 |
337 |
663 |
33.70 |
2000 |
3 |
0.15 |
|
980 |
447 |
553 |
44.70 |
2000 |
2 |
0.10 |
|
965 |
459 |
541 |
45.90 |
2000 |
0 |
0.00 |
|
240 |
988 |
338 |
662 |
33.80 |
2000 |
0 |
0.00 |
986 |
399 |
601 |
39.90 |
2000 |
0 |
0.00 |
|
975 |
436 |
564 |
43.60 |
2000 |
3 |
0.15 |
|
966 |
375 |
625 |
37.50 |
2000 |
0 |
0.00 |
|
984 |
460 |
540 |
46.00 |
2000 |
0 |
0.00 |
|
971 |
427 |
573 |
42.70 |
2000 |
0 |
0.00 |
|
480 |
982 |
172 |
828 |
17.20 |
2000 |
3 |
0.15 |
962 |
371 |
629 |
37.10 |
2000 |
2 |
0.10 |
|
969 |
399 |
601 |
39.90 |
2000 |
7 |
0.35 |
|
994 |
152 |
848 |
15.20 |
2000 |
3 |
0.15 |
|
974 |
230 |
770 |
23.00 |
2000 |
1 |
0.05 |
|
991 |
271 |
729 |
27.10 |
2000 |
4 |
0.20 |
|
CPA, 20+ |
970 |
297 |
703 |
29.70 |
2000 |
34 |
1.70 |
993 |
219 |
781 |
21.90 |
2000 |
18 |
0.90 |
|
996 |
339 |
661 |
33.90 |
2000 |
22 |
1.10 |
|
976 |
352 |
648 |
35.20 |
2000 |
22 |
1.10 |
|
964 |
257 |
743 |
25.70 |
2000 |
13 |
0.65 |
|
967 |
188 |
812 |
18.80 |
2000 |
31 |
1.55 |
+ administered as a single dose; MN micronucleated
Table 1: Induction of unscheduled DNA synthesis by chloroform and positive controls in the hepatocyte DNA repair assay.
Chemical |
Dose (mg/kg) |
Time (hr) |
Number of animals |
NG ± SE a) |
%IR ± SE b) |
2-AAF c) |
50 |
2 |
4 |
13.2 ± 2.7 |
71 ± 7 |
12 |
3 |
45.0 ± 11.3 |
96 ± 3 |
||
DMN d) |
10 |
2 |
4 |
55.8 ± 3.3 |
91 ± 4 |
MNNG e) |
50 |
2 |
3 |
1.2 ± 3.5 |
21 ± 10 |
Chloroform c) |
40 |
2 |
3 |
-4.1 ± 0.4 |
2 ± 1 |
400 |
2 |
3 |
-4.4 ± 0.8 |
1 ± 1 |
|
12 |
3 |
-2.7 ± 0.3 |
4 ± 1 |
||
Corn oil |
2 |
7 |
-5.1 ± 0.5 |
1 ± 0 |
|
12 |
13 |
-4.4 ± 0.5 |
3 ± 1 |
||
Water |
2 |
3 |
-4.8 ± 0.8 |
6 ± 2 |
|
12 |
4 |
-4.3 ± 0.5 |
1 ± 1 |
||
DMSO |
2 |
3 |
-3.0 ± 1.7 |
5 ± 4 |
|
12 |
3 |
-3.3 ± 0.6 |
2 ± 1 |
a) NG is net grains/nucleus; standard errors shown represent animal-to-animal variation; b) %IR is percentage of cells with ≥ 5 NG; c) Administered in corn oil by oral gavage, controls received corn oil; d) Administered in water by oral gavage, controls received water; e) Administered in DMSO by intraperitoneal injection; controls received DMSO ip; 2-AAF: 2-acetylaminofluorene; B(a)P: Benzo(a)pyrene; DMN: Dimethylnitrosamine; MNNG: N’-methyl-N’-nitro-N-nitrosoguanidine; DMSO: Dimethylsulfoxide
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro studies:
Bacterial Mutagenicity
Araki (2004) report a bacterial reverse mutation assay on carbon tetrachloride (TCM) using Salmonella strains TA 1535, TA 1537, TA 98 and TA 100 andE. colistrains WP2 uvrApKM101 and WP2 pKM 101 exposed to TCM using the gas exposure method. A concentration dependent rise of the number of revertants was seen in strains TA98, WP2uvrA pKM101 and WP2 pKM 101. CTC is considered to be positive in the bacterial mutagenicity test.
BASF (1985) report a bacterial reverse mutation assay on 1,2 -Dichloroethane (DCE) using Salmonella strains TA 1535, TA 1537, TA 98 and TA 100 exposed to DCE using the plate incoporation and dessciator jar methods. A concentration dependent rise of the number of revertants was seen in strains TA1535 abd TA100. DCE is considered to be positive in the bacterial mutagenicity test.
Araki (2004) report a bacterial reverse mutation assay on chloroform using Salmonella strains TA 1535, TA 1537, TA 98 and TA 100 andE. colistrains WP2 uvrApKM101 and WP2 pKM 101 exposed to chloroform using the gas exposure method. No increase in the number of revertants was seen in strains TA98, WP2uvrA pKM101 and WP2 pKM 101. Chloroform is considered to be negative in the bacterial mutagenicity test.
In Vitro Mammalian Cell Genotoxicity - Cytogenicity
Garry (1990, reported in the ATSDR report of 2005), reports a chromosome aberration study in human lymphocytes in vitro. Lymphocytes were exposed to TCM, both in the presence and absence of S9. The dose levels of TCM included a level that was toxic, confirming that the dose range of the study was valid. No increases in chromosome aberrations was observed after exposure to TCM and TCM was concluded to be negative for chromosome damage.
Gregg (1993) reports a chromosome aberration study in Chinese hamster lung cell (CHL) in vitro. CHL cells were exposed to DCE, both in the presence and absence of S9. The dose levels of DCE included a level that was toxic, confirming that the dose range of the study was valid. No increases in chromosome aberrations was observed after exposure to DCE in the absence of S9, but dose-related increases in the frequency of chromosome aberrations were observed in the presence of S9. DCE was concluded to be positive for chromosome damage.
Kirkland (1981), reports a chromosome aberration study in human lymphocytes in vitro. Lymphocytes were exposed to chloroform, both in the presence and absence of S9. The dose levels of chloroform included a level that was toxic, confirming that the dose range of the study was valid. No increases in chromosome aberrations was observed after exposure to chloroform and chloroform was concluded to be negative for chromosome damage.
In Vitro Mammalian Cell Genotoxicity - Mutagenicity
Tafazoli (1998, reported in the ATSDR report of 2005), reports a alkaliine gel electrophoresis (COMET) study in human lymphocytes in vitro. Lymphocytes were exposed to TCM, both in the presence and absence of S9. The dose levels of TCM included a level that was toxic, confirming that the dose range of the study was valid. Increases in DNA damage was observed after exposure to TCM and TCM was concluded to be positive for mutagenicity.
Tan (1985) reports a gene mutation (HGPRT) study in Chinese hamster ovary cells (CHO) in vitro. CHO cells were exposed to DCE, both in the presence and absence of S9. The dose levels of DCE included a level that was toxic, confirming that the dose range of the study was valid. Increases in the frequency of mutations was observed after exposure to DCE in the absence and presence of S9. DCE was concluded to be positive for mutagenicity.
Muller (2004) reports a gene mutation (HGPRT) study in Chinese hamster cells (V79) in vitro. V79 cells were exposed to chloroform, both in the presence and absence of S9. The dose levels of chloroform included a level that was beyond the solubility limit, confirming that the dose range of the study was valid. No toxicologically significant Increases in the frequency of mutations was observed after exposure to chloroform in the absence or presence of S9. Chloroform was concluded to be negative for mutagenicity.
In vivo studies:
In Vivo Mammalian Cell Genotoxicity - Cytogenicity
Suzuki (1997), reports a mouse micronucleus test. Administrations of carbon tetrachloride (TCM) were made once or twice (24 hours apart) by gavage and sampling 24 hours later for the bone marrow assay, or once by the ip route with samplings after 0, 24, 48 and 72 hours for the peripheral blood assay. In none of the investigations were the micronucleated polychromatic erythrocytes (bone marrow) or micronucleated reticulocytes (peripheral blood) increased at any time point. TCM has no mutagenic potential in this test system.
Armstrong (1993) reports that DCE was examined for its possible cytogenetic effects (induction of chromosomal damage) using the micronucleus test, similarily conducted according to OECD Test Guideline 474 (Mammalian Erythrocytes Micronucleus Test) . Micronucleus (MN) induction in peripheral blood was examined in 10 male and female mice (lymphoma prone Eµ - PIM 1 transgenic mice) per dose after a repeated dose of 1,2 -dichloroethane in corn oil administered by gavage (5 mL/kg bw) once a day in concentrations of 100 and 200 mg/kg bw (in males) and 150 and 300 mg/kg bw (in females) up to week 6. After week 6 until the end of the exposure period (week 41) the top doses were reduced, therefore male animals were only administered with 100 mg/kg bw and females were only administered with 150 mg/kg bw (due to mortality and treatment related weight gain). Two negative controls were performed, a vehicle control and a “true” negative control with methyl cellulose. Peripheral blood was collected at week 14 and at terminal sacrifice (41 week) and analysed a week later (week 42). Stained slides (acridine orange staining) from 10 animals per treatment group were analysed for the number of micronucleated cells per 1000 NCE (normochromatic erythrocytes) and the number of PCE (polychromatic erythrocytes) per 1000 erythrocytes (Since exposure was discontinued one week before the terminal harvest of mice, only NCE were scored for micronuclei because of the short residence time of polychromatic erythrocytes. Results showed no micronucleus induction (in normochromatic erythrocytes) after 14 weeks or 41 weeks of 1,2 -dichloroethane exposure and no polychromatic erythrocyte suppression was detected in the blood after 14 weeks or 41 weeks of exposure to the test substance. It can therefore be concluded that under the study conditions, 1,2 -dichloroethane induced no cytogenetic damage/chromosome damage.
Whitwell (2009), reports that chloroform was tested for its ability to induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female young adult Sprague Dawley rats, following 5 days of repeat oral dosing. The test was carried out under GLP conditions and in accordance with OECD Guideline No. 474. The micronucleus study was performed by administration by oral gavage of 120, 240 or 480 mg/kg body weight/day chloroform in corn oil for five consecutive days to groups of six male and six female rats, respectively. The group mean frequency of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE), ratio expressed as % PCE, of the negative vehicle controls were considered as valid, although negative male control rats exhibited a slightly increased ratio. The positive control groups exhibited significantly increased frequencies of micronucleated PCE in comparison with the concurrent controls. The assay system was therefore considered as valid. Rats treated with chloroform showed group mean % PCE values that decreased in a dose dependent manner, with the highest dose group (480 mg/kg/day) exhibiting 38 % PCE (males) or 27 % PCE (females). These were markedly lower than the concurrent vehicle control values of 62 % or 44 % PCE (male and females respectively) and also lower than historical values and as such were considered to represent some evidence of bone marrow toxicity and target organ exposure. The group mean frequencies of micronucleated PCE observed in the groups treated with chloroform were not significantly different to the vehicle controls. In addition, individual frequencies of micronucleated PCE were generally similar to those seen in the vehicle control groups and consistent with the laboratory's historical control distribution data. From the results of the study it is concluded that chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male and female Sprague Dawley rats treated up to 480 mg/kg body weight/day for five consecutive days, under the experimental conditions employed. Thus, chloroform was not genotoxic in the micronucleus assay.
In Vivo Mammalian Cell Genotoxicity - Mutagenicity
Mirsalis (1982), reports a study on unscheduled DNA synthesis in Fisher 344 rat liver cells. Rats were exposed to TCM by oral gavage at 40 and 400mg/kg. The animals were sampled after 2 or 12 hours post-dosing and the liver cells examined for unscheduled DNA synthesis, which is taken as evidence for DNA damage. No evidence of DNA damage was detected.
Haychiya (2000) reports on the induction of lacZ mutation in the liver and testis of Muta™Mouse following the administration of DCE. No increase was detected in the frequency following DCE administration of single doses of up to 150 mg/kg, or after consecutive injections of up to 280 mg/ kg. The results indicate that DCEis negative for the induction of gene mutation in the liver of Muta™Mouse.
Mirsalis (1982), reports a study on unscheduled DNA synthesis in Fisher 344 rat liver cells. Rats were exposed to chloroform by oral gavage at 40 and 400mg/kg. The animals were sampled after 2 or 12 hours post-dosing and the liver cells examined for unscheduled DNA synthesis, which is taken as evidence for DNA damage. No evidence of DNA damage was detected.
Justification for classification or non-classification
The classification for mutagenicity is based on the rules of the CLP Regulation for classification of mixtures.
Harmonized classification:
The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).
Self-classification:
None of the three constituents has genotoxic potential in vivo and consequently classification is not required because it is anticipated that the registered substance will also show no genotoxic potential in vivo.
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