Chloroform

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Use of primary cultures of human hepatocytes in toxicology studies

Author:

Butterworth BE, Smith-Oliver T, Earle L, Loury DJ, White RD, Doolittle DJ, Working PK, Cattley RC, Jirtle R, Michalopoulos G, & Strom S

Year:

1989

Bibliographic source:

Cancer Res, 49: 1075-1084

Materials and methods

Principles of method if other than guideline:

Butterworth et al. (1987): Mutat. Res. 189, 113-121.
Strom et al. (1982): J. Natl. Cancer Inst. 68, 771-778.
Butterworth et al. (1983): Mutat. Res. 122, 73-80.
Mirsalis et al. (1982): Environ. Mutagen. 4, 553-562.
Briefly, the hepatocytes were incubated in media containing the test chemical and 10 µci/ml [3H] thymidine.

GLP compliance:

no

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Chloroform

EC Number:

200-663-8

EC Name:

Chloroform

Cas Number:

67-66-3

Molecular formula:

CHCl3

IUPAC Name:

trichloromethane

Details on test material:

Test substance: chloroform
Source: Fischer (Raleigh, NC)
Batch: no data
Purity: ACS certified

Method

Species / strain

Species / strain / cell type:

primary culture, other: human hepatocytes

Details on mammalian cell type (if applicable):

Fresh human tissue was obtained as excess material from prescribed surgery. Small portions of apparently healthy tissue not need for pathological examination were used. Catheters were inserted in the larger vesselson the cut surface, the tissue was perfused with collagenase solution, and a primary hepatocyte culture was established.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations with justification for top dose:

0, 0.01, 0.1 and 1.0 mM

Vehicle / solvent:

DMSO (1 %) added to 10 uCi/mL [3H]-thymidine

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO

True negative controls:

yes

Positive controls:

no

Positive control substance:

no

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-acetylamino-fluorene (2-AAF), Aflatoxin B1, 2-aminobenzyl alcohol (2-ABA), Benzo(a)pyrene, Dimethylnitrosamine (DMN)

Details on test system and experimental conditions:

Human Hepatocyte Preparation: Fresh human tissue was obtained as excess material from prescribed surgery. Small portions of apparently healthy tissue not needed for pathological examination were placed in ice cold saline and transported to the laboratory. Catheters were inserted in the larger vessels on the cut surface, the tissue was perfused with a collagenase solution, and a primary hepatocyte culture was established as described previously (Strom et al., (1982) J.Natl. Cancer Inst., 68, 771-778). UDS Experiments: Induction of DNA repair in the human cultures was performed as described previously (Butterworth et al. (1983) Mut. Res., 122, 73-80). Briefly, the hepatocytes were incubated in media containing the test chemical and 10 µCi/ml [3H]thymidine.
Autoradiography and Evaluation of Results: Slides were air dried, dipped in NTB 2 photographic emulsion (Kodak, Rochester, NY) diluted 1:1 with water and exposed for 8 days at 20°C. Slides were developed and scored. Silver grains over the nucleus minus the grains over an equal sized area in the cytoplasm was defined as net grains per nucleus and quantitated with an automatic grain counter. A negative number indicates there were more grains per unit area in the cytoplasm than in the nucleus. As a conservative estimate, any individual cell with greater than or equal to 5 NG was considered in repair. The percentage of cells in repair was also calculated as an indication of the extent of the response among the cells. Historical observations with rat hepatocytes indicate that if a chemical induces greater than or equal to 5 NG (population average) and greater than or equal to 20% of cells in repair, the response can be considered positive. A population average between 0 NG and 5 NG would be considered a marginal response. Because human samples differ so much from each other it was not possible to establish tight historical controls or strict criteria to define a positive response. Control cells from the same preparation do represent a true concurrent control.

Evaluation criteria:

Any individual cell with greater than or equal to 5 NG was considered in repair. The percentage of cells in repair was also calculated as an indication of the extent of the response among the cells. Historical observations with rat hepatocytes indicate that if a chemical induces greater than or equal to 5 NG (population average) and greater than or equal to 20 % of cells in repair, the response can be considered positive.

Statistics:

Control cells from the same preparation do represent a true concurrent control. The unpaired t test for the equality of two means with the NG counts (population average) of the individual slides as the unit of measure is an appropriate statistical test for these data.

Results and discussion

Test results

Key result

Species / strain:

other: human hepatocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

NG values for media control were -5.5 +/- 5.1 (case 12), -1.0 +/- 4.3 (case 13) and -6.4 +/- 5.7 (case 14); NG values for solvent control (1 % DMSO) were -2.7 +/- 4.7, -0.3 +/- 4.4, and -5.8 +/- 4.4, respectively. NG values resulting from exposure to chloroform of case 12 at 0.1-1.0 mM ranged from -4.8 to -6.8, NG values resulting from exposure to chloroform of case 13 at 0.01-1.0 mM ranged from -0.1 to -1.8, and NG values resulting from exposure to chloroform of case 14 at 0.01-1.0 mM ranged from -5.5 to -9.8. NG values resulting from exposure to DMN at 0.01 to 1.0 mM of cas 12, 13 and 14 ranged from 2.8 to 31.6, 17.2 to 30.8 and from 20.1 to 26.1, respectively.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: DNA repair response

Chemical	Concentration (mM)	Case 12		Case 13		Case 14		Case 15
--	--	NG ± SD a)	%IR b)	NG ± SD a)	%IR b)	NG ± SD a)	%IR b)	NG ± SD a)	%IR b)
Media control	--	-5.5 ± 5.1	2	-1.0 ± 4.3	8	-6.4 ± 5.7	2	-3.9 ± 3.9	0
Solvent control	1 % DMSO	-2.7 ± 4.7	3	-0.3 ± 4.4	12	-5.8 ± 4.4	1	-4.1 ± 5.5	5
2-AAF	0.001	--	--	21.6 ± 11.3 c)	99	1.3 ± 7.4 c)	32	-1.2 ± 3.6	5
	0.01	--	--	33.3 ± 15 c)	99	5.6 ± 7.8 c)	47	0.9 ± 3.9 c)	15
	0.1	28.8 ± 12.1 c)	100	33.0 ± 11.2 c)	100	2.8 ± 7.7 c)	39	Toxic e)	--
Aflatoxin B1	0.0001	12.3 ± 16.7 c)	60	15.6 ± 9.0 c)	88	16.4 ± 9.3 c )	91	5.9 ± 8.0 c)	54
	0.001	9.1 ± 13.4 c)	58	24.9 ± 13.1 c)	97	59.5 ± 21.0 c)	100	12.3 ± 6.4 c)	91
	0.01	31.0 ± 11.9 c)	100	34.6 ± 16.3 c)	99	61.8 ± 20.4 c)	100	129.7 ± 74.7 c)	100
2-ABA	0.01	33.5 ± 14.5 c)	100	64.8 ± 21.6 c)	100	7.0 ± 8.7 c)	57	--	--
	0.1	25.0 ± 9.7 c)	99	63.8 ± 30.8 c)	100	89.8 ± 30.4 c)	100	--	--
	1.0	50.7 ± 19.3 c)	100	56.6 ± 18.6 c)	100	94.9 ± 35.6 c)	100	--	--
Benzo(a)pyrene	0.001	--	--	11.5 ± 6.7 c)	87	18.7 ± 9.4 c)	94	Toxic f)	--
	0.01	--	--	21.6 ± 11.4 c)	95	11.8 ± 8.8 c)	83	Toxic f)	--
	0.1 d)	--	--	18.5 ± 9.1 c)	98	26.5 ± 11.6 c)	97	8.6 ± 12.7 c)	70
DMN	0.1	2.8 ± 7.1 c)	37	17.2 ± 9.3 c)	94	20.1 ± 10.3 c)	97	15.5 ± 18.0 c)	78
	1.0	27.8 ± 13.8 c)	96	33.2 ± 14.9 c)	99	24.2 ± 13.7 c)	96	53.8 ± 35.3 c)	91
	10	31.6 ± 17.9 c)	93	30.8 ± 13.3 c)	98	26.1 ± 13.4 c)	95	42.2 ± 19.5 c)	100
Chloroform	0.01	--	--	-1.8 ± 4.3	5	-5.5 ± 4.2	1	-2.1 ± 3.7	5
	0.1	-4.8 ± 5.0	3	-0.4 ± 3.5	6	-8.0 ± 7.5	1	-2.3 ± 3.8	3
	1.0	-6.8 ± 5.3	1	-0.1 ± 3.3	5	-9.8 ± 7.4	1	-3.1 ± 4.2	1

a) One hundred fifty cells were counted on one slide. SD is cell-to-cell variation; b) An individual cell with ≥ 5 NG was considered in repair (IR); c) Greater than the average of the cells on the solvent control slide by the unpaired t test for the equality of two means at p ≤ 0.05; d) Solubility exceeded; e) Difficult to evaluate because cytoplasms were missing; f) Cells pyknotic and sparse with few grains.

Applicant's summary and conclusion

Conclusions:

In the in vitro DNA damage and repair assay (unscheduled DNA synthesis) in human hepatocytes, there was a negative response after exposure to chloroform at 0.01, 0.1 and 1.0 mM.

Executive summary:

A test of induction of DNA repair in human hepatocytes was performed with chloroform at concentrations of 0.01, 0.1 and 1.0 mmol. None of the tested concentrations produced a genotoxic response in the human hepatocytes. Positive controls, in contrast, produced a positive response in the human hepatocytes and therefore the test was considered as valid. In the in vitro DNA damage and repair assay (unscheduled DNA synthesis) in human hepatocytes, there was a negative response after exposure to chloroform at 0.01, 0.1 and 1.0 mM. Chloroform was not mutagenic in this in vitro assay.