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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

In vitro gene mutation study in bacteria:

Chloral hydrate was determined to induce mutations in Salmonella typhimurium TA 100 whwereas unable to induce in strains TA 98 and TA 1535 with and without rat liver microsomal S9 metabolic activation system.

Chromosome aberration:

Chloral hydrate induced significant increases in chromosomal aberrations in cultured Chinese hamster ovary cells, with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.

Based on different available data for genetic toxicity in vitro, chloral hydrate is observed to be ambiguous in nature (i.e. both positive and negative). However considering the Harmonised classification of target, it can be concluded that the substance Chloral hydrate is non mutagenic in nature.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Chloral hydrate was screened for mutagenicity in the Salmonella/rat-liver microsomal assay system developed by Dr. B. Ames et al.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material: 2,2,2-Trichloroethane-1,1-diol (Chloral hydrate)
- Molecular formula: C2-H3-Cl3-O2
- Molecular weight: 165.4026g/mol
- Smiles notation: C(C(O)O)(Cl)(Cl)Cl
- InChl: RNFNDJAIBTYOQL-UHFFFAOYSA-N
- Substance type: Organic
- Physical state: Solid
Species / strain / cell type:
S. typhimurium, other: TA98, TA 100 and TA 1535
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomal S9 metabolic activation system.
Test concentrations with justification for top dose:
0.5 - 10 mg (maximum non-toxic dose)
Vehicle / solvent:
not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Bacteria 0.1 ml of a stationary culture of Salmonella typhimurium strains in broth, 60 ƛ of microsomes and a NADPH-generating system (5 µmoles glucose 6-phosphate, 4 µmoles NADP, 1 unit glucose-6-phosphate dehydrogenase) were mixed in a final volume of 1 ml in either 0.1 M sodium phosphate buffer, pH 7.4 or 0.05 M Tris-HCl buffer pH 7.4 at 37°C. The anesthetic concentration in the final reaction mix was 2 mM. This reaction mixture was incubated in a sealed 5-ml vial for 30 min at 37°C before being added to the top agar and plated.
Rationale for test conditions:
not specified
Evaluation criteria:
Number of his+ revertants above control
Statistics:
Student’s t test
Key result
Species / strain:
S. typhimurium, other: TA 98 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
not specified
Conclusions:
Chloral hydrate was determined to induce mutations in Salmonella typhimurium TA 100 whwereas unable to induce in strains TA 98 and TA 1535 with and without rat liver microsomal S9 metabolic activation system.
Executive summary:

Chloral hydrate was screened for mutagenicity in the Salmonella/rat-liver microsomal assay system developed by Dr. B. Ames et.al. (Standard plate incorporation assay of Ames test). The study was performed using Salmonella typhimurium strains TA98, TA100 and TA1535 in the presence and absence of rat liver microsomal S9 metabolic activation system. The test chemical was used at dose levels of 0.5 to 10 mg/plate. The number of his+ revertants above control was evaluated for each strain. The test material did not exhibit induction of his+ revertants in Salmonella typhimurium TA98 and TA1535, either with or without rat liver microsomal S9 metabolic activation system. While in strain TA100 it did induces the number of revertants with statistically significant result by Student’s t test with p values of <0.01, both with and without metabolic activation system.

Thus the substance Chloral hydrate is determined to be ambiguous in nature for in vitro genetic toxicity test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from NTP technical report
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Chloral hydrate was tested in cultured Chinese hamster ovary (CHO) cells for chromosomal aberrations (Abs), both in the presence and
absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.
GLP compliance:
not specified
Type of assay:
other: chromosome aberration test
Specific details on test material used for the study:
- Name of test material: 2,2,2-Trichloroethane-1,1-diol (Chloral hydrate)
- Molecular formula: C2-H3-Cl3-O2
- Molecular weight: 165.4026g/mol
- Smiles notation: C(C(O)O)(Cl)(Cl)Cl
- InChl: RNFNDJAIBTYOQL-UHFFFAOYSA-N
- Substance type: Organic
- Physical state: Solid
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.
Test concentrations with justification for top dose:
750, 1000, 1250, 1500 μg/mL – without S9
2,500, 3000, 3500 μg/mL – with S9
(highest non toxic dose)
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
In the Abs test without S9, cells were incubated in McCoy’s 5A medium with chloral hydrate for 18.5 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with chloral hydrate and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 10.5 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test: because cell cycle delay was anticipated in the absence of S9, the incubation period for these cultures was extended.
Rationale for test conditions:
not specified
Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred first-division metaphase cells were scored at most dose levels; occasionally, when a high percentage of aberrant cells was present in the culture, fewer cells were scored. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized
cells, despiralized chromosomes, and cells containing 10 or more aberrations).
Statistics:
Chromosomal aberration data are presented as the percentage of cells with aberrations. To arrive at a statistical decision, analyses were conducted on both the dose-response curve and individual dose points. For a single trial, a statistically significant (P#0.05) difference for one dose point and a significant trend (P≤0.015) was considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend, in the absence of a statistically significant increase at any one dose resulted in an equivocal response. Ultimately, the trial responses were based on a consideration of the statistical analyses as well as the biological information available to the reviewers.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Chloral hydrate induced significant increases in chromosomal aberrations in cultured Chinese hamster ovary cells, with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.
Executive summary:

Chloral hydrate was tested in cultured Chinese hamster ovary (CHO) cells for chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. The test substance concentration used in the study on second trial of experiment were 750, 1000, 1250, 1500 μg/mL for without S9 activation and 2,500, 3000, 3500 μg/mL with S9 activation system. The substance was dissolved in DMSO and it also served as vehicle control. The positive control used was Cyclophosphamide for with S9 system and Mitomycin-C without S9 system. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Chloral hydrate was found to induce significant increases in chromosomal aberrations in cultured Chinese hamster ovary cells, with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

No. of studies of Chloral hydrate reviewed for genetic toxicity from reliable sources having Klimisch rating 2. The summary of the results are presented below: 

In key study by Lucy Waskell in Mutation Research, 57 (1978) 141-153, Chloral hydrate was screened for mutagenicity in the Salmonella/rat-liver microsomal assay system developed by Dr. B. Ames et.al. (Standard plate incorporation assay of Ames test). The study was performed using Salmonella typhimurium strains TA98, TA100 and TA1535 in the presence and absence of rat liver microsomal S9 metabolic activation system. The test chemical was used at dose levels of 0.5 to 10 mg/plate. The number of his+ revertants above control was evaluated for each strain. The test material did not exhibit induction of his+ revertants in Salmonella typhimurium TA98 and TA1535, either with or without rat livermicrosomal S9 metabolic activation system. While in strain TA100 it did induces the number of revertants with statistically significant result by Student’s t test with p values of <0.01, both with and without metabolic activation system.

Thus the substance Chloral hydrate is determined to be ambiguous in nature for in vitro genetic toxicity test.

While in a NTP Technical Report on the Toxicity and Metabolism Studies of Chloral Hydrate by Frederick A. Beland (1999), chloral hydrate was tested in cultured Chinese hamster ovary (CHO) cells for chromosomal aberrations (Abs), both in the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix. The test substance concentration used in the study on second trial of experiment were 750, 1000, 1250, 1500 μg/mL for without S9 activation and 2,500, 3000, 3500 μg/mL with S9 activation system. The substance was dissolved in DMSO and it also served as vehicle control. The positive control used was Cyclophosphamide for with S9 system and Mitomycin-C without S9 system. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Chloral hydrate was found to induce significant increases in chromosomal aberrations in cultured Chinese hamster ovary cells, with and without Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix.

Further in Mutagenesis vol. 26 no. 6 pp. 771–781, 2011, Mouse lymphoma TK assay was performed to detect the genotoxic nature of the test material chloral hydrate using 24 hrs exposure period in mouse lymphoma L5178Y Tk+/–cells, clone 3.7.2c in the absence of an exogenous metabolic activation system. 4-NQO was used as the positive control. In the study performed, increased mutant frequency was seen only at high levels of toxicity. Toxicity was measured by reduction in relative total growth (RTG), which is the product of suspension growth and Day 2 cloning efficiency. Although some evidence of mutagenicity was seen with chloral hydrate, the increases were generally at levels of survival, 10% and it is likely that, if tested as unknown compounds, the majority of these results would not be considered positive using currently accepted mouse lymphoma assay evaluation criteria. None of the chemicals tested would be unequivocally classified as positive using currently accepted criteria.Gene toxicity in vitro result for the test material Chloral hydrate is negative upto a concentration of 2700 µmol/L. Thus, the test material chloral hydrate is not likely to classify as a gene mutant.

Whereas data obtained from Mutation Research 334 (1995) 1-7 indicates that Identification of aneuploidy is becoming an important phase of genotoxicity evaluation. Aneuploidy-inducing agents generally induce micronuclei (MN) containing whole chromosome(s) or chromatids. The in vitro micronucleus assay, as a rapid screen for genotoxic activity, is useful to investigate this type of compound. The cytochalasin B micronucleus test was performed in human peripheral lymphocyte cultures to assay the ability chloral hydrate to induce micronuclei, in the presence or absence of an exogenous metabolic activation system. Colchicine, a well-known spindle poison, was selected as the positive control and the controls used were DMSO and distilled water+ S9.

 Cultures and readings were performed in duplicate. 1000 binucleated lymphocytes (CB) were examined per culture for the presence of micronuclei (MNCB). In addition, the percentages of mononucleated, binucleated, polynucleated and mitotic cells were recorded. On treatment with chloral hydrate, only treatment without metabolic activation resulted in a dose-dependent decrease in the mitotic ratio. A dose-dependent increase in the MNCB frequency was not observed with any of the treatments. Positive control compound colchicine showed statistically significant induction of micronuclei. Since no significant damage was found after treatment with chloral hydrate in this test system hence Chloral hydrate is negative for gene toxicity invitro by cytochalasin B / human lymphocyte micronucleus test.

Further In vitro micronucleus assay was carried out using L5178Y TK+/– clone 3.7.2C mouse lymphoma cellsto enable rapid screening of chloral hydrate for its genotoxicity potential ( Mutagenesis. 1999 Jul;14(4):403-10.). The test was performd in two ways-

1) In treatment without metabolic activation system (24h – S9), cells were continuously treated for 24 hrs.

2) In treatment without metabolic activation system (24h + 20 h – S9), cells were continuously treated for 24 hrs. After this treatment, the test material was discarded and the cells were washed in FM10. The cells were re-suspended in FM10 and were further incubated for 20h.

According to both the methods,a6 fold increase in the number of MN over the control is observed. Hence the in vitro genetic toxicity and cytotoxicity of chloral hydrate is found to be positive.

Based on the above available data for genetic toxicity in vitro, chloral hydrate is observed to be ambiguous in nature (i.e. both positive and negative). However considering the Harmonised classification of target, it can be concludedthat the substance Chloral hydrate is non mutagenic in nature.

Justification for classification or non-classification

Based on the above available data for genetic toxicity in vitro, chloral hydrate is observed to be ambiguous in nature (i.e. both positive and negative). However considering the Harmonised classification of target, it can be concludedthat the substance Chloral hydrate is non mutagenic in nature.