Chloroform

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The majority of in vitro assays on the mutagenicity of chloroform produced negative results. Only in a few studies slightly positive results were observed. 

Link to relevant study records

Reference

Reference 1

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

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

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO

True negative controls:

yes

Positive controls:

no

Positive control substance:

no

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.

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

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.

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.

Reference 2

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:

guideline study with acceptable restrictions

Principles of method if other than guideline:

Gas phase exposure using a gas sampling bag (Araki, A., Noguchi, T., Kato, F., and Matsushima, T. 1994. Mutat. Res. 307, 335-344.)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix (4mM NADPH, 4mM NADH, 5mM G-6-P, 8mM MgCl2, 33mM KCl, 100mM sodium phosphate buffer pH 7.4, 10% S9); S9 Mix with additional glutathione (10mM GSH)

Test concentrations with justification for top dose:

0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0 % in the gas phase

Vehicle / solvent:

Vehicle/solvent: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: air only

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Remarks:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2furyl)acrylamide

Remarks:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

no

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, B.N., McCann, J., and Yamasaki, E. 1975. Mutat. Res. 31, 347-364) was used for determining mutagenicity in individual experiments.

Statistics:

To compare the response for a particular dose group in a particular tester strain (in some cases more than three data values per dose group from different experiments) with those of the concurrent control group, Bartlett's test was first used to determine whether the variance was homogeneous or not. If the variance was homogeneous, one-way ANOVA was applied. Else, the Kruskal-Wallis rank sum test was performed, arranging all data for control and dose groups in descending order. Statistical differences in means and rank means among the groups then were analyzed by Dunnett's multiple comparison test, and the same multiple comparison test by rank, respectively. Two-sided analyses were performed, with the p-values for significance set at 0.05 and 0.01.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 5.0%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Chloroform was not mutagenic in Salmonella typhimurium strains TA98, TA 100, TA 1535, or TA 1537, with or without S9 mix, and no mutagenicity was observed in TA98, TA 100, TA 1535, TA 1537 in the presence of glutathione-supplemented S9 mix.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Exposure of Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 to chloroform via the gas phase did not cause mutagenicity up to a dose of 2 %.

Executive summary:

The present study investigated the mutagenicity of chloroform in Salmonella typhimurium strains TA98, TA100, TA1535, and TA 1537 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 TA98, TA100, TA1535 or TA1537, with or without S9 mix, and no mutagenicity was observed in TA98, TA100, TA1535 or TA1537 in the presence of glutathione-supplemented S9 mix.

Reference 3

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:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Ames et al 1975

Principles of method if other than guideline:

Method: Ames et al (1975) Mut Res, 31, 347

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: E. coli WP2p

Additional strain / cell type characteristics:

other: tryptophan auxotroph and carrying the Ampillicin resistance plasmid, fully DNA-repair proficient

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan auxotroph and carrying the Ampillicin resistance plasmid, deficient in excision repair at the uvrA locus

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 0.1, 1, 10, 100, 1000, 10000 µg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Remarks:

no

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

1 and 10 ug/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

5 and 50 ug/plate

Details on test system and experimental conditions:

Two strains of E. coli, WP2p and WP2uvrA-p were treated with chloroform in plate incorporation and pre-incubation tests both with and without rat-liver microsomes (S-9) prepared from Aroclor 1254-induced PVG/Ola Hooded rats. In each case chloroform was administered at 10,000, 1000, 100, 10, 1 or 0.1 ug/plate using acetone as the diluent. The method was essentially that of Ames et al. (1975) for S. typhimurium in that to 2 ml of molten top agar at 45°C were added 0.1 ml of a dilution of chloroform plus 0.1 ml (approximately 10e8 organisms) of an overnight broth culture of the tester bacteria, and, where appropriate, 0.5 ml of S-9 mix containing 10%, S-9 with standard co-factors (Ames et al. 1975). These ingredients were rapidly mixed and poured onto prepared Vogel-Bonner agar plates. Each treatment was carried out in triplicate, and the three plates for each dose either with or without S-9 were separately packed in gas-tight containers to avoid leakage of the volatile substances. The plates were incubated at 37°C for 48 hr, after which revertant colonies were counted on a Biotran II automatic colony counter.
Negative controls were included in each experiment by replacing the chloroform with acetone, and positive controls were also included; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1 or 10 µg/plate) without S-9 mix, and 2-aminoanthracene (2AA, 5 or 50 µg/plate) with 10% S-9 mix. The test was repeated on separate days with fresh cultures, solutions and controls.

Evaluation criteria:

Abnormalities were classified as previuosly reported (Kirkland et al, 1978) and using the system of Bauchinger et al, 1976

Statistics:

not reported

Species / strain:

E. coli, other: WP2p and WP2uvrA-p

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1: Plate incorporation assay of chloroform and positive control chemicals using Escherichia coli (values are means for three plates)

--		Mean number of revertants/plate in strain
		WP2p		WP2uvrA-p
Chemical	Amount (mg/plate)	Without S9 mix	With 10 % S9 mix	Without S9 mix	With 10 % S9 mix
Experiment 1
Chloroform	0	8.7	5.0	5.3	4.0
	0.1	7.7	7.0	6.3	1.7
	1	11.0	4.7	6.3	3.7
	10	11.3	8.7	5.3	3.0
	100	8.3	3.7	7.0	8.3
	1000	5.0	4.0	16.7	4.0
	10000	9.0*	4.7*	6.3*	3.3*
MNNG	0	6.7	--	6.7	--
	1	57.3	--	58.5	--
	10	498.3	--	644.0	--
2AA	0	--	8.0	--	22.5
	5	--	15.3	--	289.0
	50	--	18.5	--	386.0
Experiment 2
Chloroform	0	11.7	15.0	4.3	11.3
	0.1	8.3	8.3	9.7	20.3
	1	8.0	17.7	3.0	25.3
	10	9.3	14.7	6.7	17.3
	100	10.3	17.7	7.3	15.7
	1000	6.0	21.3	6.0	13.0
	10000	16.0*	13.5*	4.0	14.0*
MNNG	0	12.0	--	14.7	--
	1	230.0	--	202.3	--
	10	1873.7	--	1091.7	--
2AA	0	--	27.7	--	51.7
	5	--	21.3	--	268.7
	50	--	34.7	--	752.3

MNNG: N-Methyl-N’-nitro-N-nitrosoguanidine; 2AA: 2-Aminoanthracene; *The background lawn was sparse or absent on some or all of the plates

Table 2: Preincubation assay of chloroform and positive control chemicals using Escherichia coli (values are means for three plates)

--		Mean number of revertants/plate in strain
		WP2p		WP2uvrA-p
Chemical	Amount (mg/plate)	Without S9 mix	With 10 % S9 mix	Without S9 mix	With 10 % S9 mix
Experiment 1
Chloroform	0	24.0	17.7	33.3	40.0
	0.1	25.7	18.3	26.7	40.3
	1	17.7	17.7	2.3*	40.5
	10	22.7	18.3	NC*	42.3
	100	NC*	16.0	NC*	44.0
	1000	NC*	19.7	NC*	52.7
	10000	NC*	NC*	NC*	NC*
MNNG	0	21.0	--	58.3	--
	1	878.0	--	455.7	--
	10	1403.3	--	1551.0	--
2AA	0	--	26.3	--	15.3
	5	--	52.0	--	152.3
	50	--	60.3	--	1239.3
Experiment 2
Chloroform	0	16.7	46.0	45.0	77.3
	0.1	21.3	32.3	48.7	60.0
	1	19.3	19.7	47.3	113.7
	10	22.0	27.7	53.0	56.3
	100	24.0	30.3	53.0	51.7
	1000	NC*	15.3*	NC*	10.7*
	10000	NC*	NC*	NC*	NC*
MNNG	0	18.3	--	84.0	--
	1	482.0	--	463.0	--
	10	2209.7	--	1672.7	--
2AA	0	--	35.7	--	139.3
	5	--	81.7	--	1360.7
	50	--	102.3	--	1990.0

MNNG: N-Methyl-N’-nitro-N-nitrosoguanidine; 2AA: 2-Aminoanthracene; *The background lawn was sparse or absent on some or all of the plates

Conclusions:

The reverse gene mutation assay of chloroform with Escherichia coli were negative up to a concentration of 10000 ug/plate.

Executive summary:

A gene mutation reversion assay was carried out with chloroform in two strains of Escherichia coli, WP2p and WP2uvrA-p. The two strains were treated with chloroform in plate incorporation and pre-incubation tests both with and without rat-liver microsomes (S9). In each case chloroform was administered at 0.1, 1, 10, 100, 1000 and 10000 microgram/plate using acetone as the diluent. Negative controls were included in each experiment by replacing the chloroform with acetone. Positive controls were also included; N-methyl-N'-nitro-N-nitrosoguanidine was used at 1 or 10 ug/plate as control without S9 -mix, and 2 -aminoanthracene was used at 5 and 50 ug/plate as control with S9 -mix. Both plate incorporation and pre-incubation tests were repeated on separate days with fresh cultures, solutions and controls. In the plate incorporation test, all counts for the treated plates fall in the usual range expected for control counts. With chloroform the pre-incubation treatment was more toxic than the plate incorporation test method, and the control counts are generally higher, but there is no evidence of mutagenic activity. All positive controls were satisfactory, but although there is variation between the experiments, both strains show better responses in the pre-incubation test controls than the controls for the incorporation test. In conclusion, the test indicated the non-mutagenicity of chloroform in the two tested strains of E. coli, both in the plate incorporation and the pre-incubation tests at tested concentrations of up to 10000 microgram/plate.

Reference 4

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

Principles of method if other than guideline:

Method: Butterworth et al., (1987) Mut Res, 189, 113-121 with minor modifications

GLP compliance:

not specified

Type of assay:

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

Species / strain / cell type:

mammalian cell line, other: Hepatocytes of Female B6C3F1 Mice

Details on mammalian cell type (if applicable):

Primary hepatocyte cultures were prepared by collagenase perfusion. Untreated mice were anaesthetised. An 18-gauge cathter was inserted into the abdominal vena cava. The liver was perfused with an EGTA solution for 1 min at 5 mL/min, then for 1.5 min at 10 mL/min, the with a solution of the collagenase enzyme for 10 min at 10 mL/min. Hepatocytes were then combed from the liver into suspension in Williams medium E (WME). Viability was assessed by Trypan blue dye exclusion. Approximately 2x10e5 viable hepatocytes were plated and allowed to attach to plastic overslips in 6-well dishes in WME with 5 % fetal bovine serum for 2-3 hours at 37 °C and 5 % CO2.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0 mM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Williams medium E

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (DMN) dimethylnitrosamine

Remarks:

no

Details on test system and experimental conditions:

Unscheduled DNA synthesis
Chemically induced DNA repair was measured in primary mouse hepatocyte cultures as described previously [Butterworth et al., 1987], with some modifications. Primary hepatocyte cultures were prepared by collagenase perfusion. For treatment, test chemicals were dissolved directly in Williams medium E containing 10 mCi/ml 3H-thymidine. Cells from each animal were incubated with 3 ml of medium containing the concentrations of chloroform for 20 hr. DMN was used as the positive control. After treatment, cells were rinsed, allowed to swell, fixed, and washed. When dry, the coverslips were mounted on microscope slides, dipped in phoytographic emulsion, and exposed in light-tight boxes at -20°C for 12 days. After developing, slides were stained with hematoxylin and eosin. Silver grains were quantitated in 25 cells for each of three duplicate slides per animal for hepatocyte preparations from each of three different animals. Net nuclear grains (NG) was calculated by counting the silver grains over the nucleus and subtracting the grain count from a nuclear-size area in the cytoplasm adjacent to the nucleus. NG are presented as a population average ± SE representing animal-to-animal variation.

Evaluation criteria:

Any individual cell with a NG value at or above 5 was considered to be in repair, and the percentage of the population in repair was also reported.

Statistics:

Statistical significance compared to controls was determined by the unpaired t-test for the equality of two means. A response was judged positive at P <= 0.05 with an NG value greater than zero.

Key result

Species / strain:

mammalian cell line, other: hepatocytes of female B6C3F1 Mice

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 10 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Table 1: UDS following in vitro exposure to chloroform or DMN in primary hepatocyte cultures from female B6C3F1 mice

Chemical	Concentration (mmol)	NG ± SE a	Percentage of cells with ≥ 5 NG
Control	Medium only	-7.5 ± 1.5	7.0 ± 4.1
Chloroform	10	Cytotoxic b	--
--	3	-12.3 ± 3.0	0.9 ± 0.4
--	1	-12.7 ± 2.7	3.5 ± 1.2
--	0.3	-12.7 ± 1.2	3.5 ± 1.6
--	0.1	-9.4 ± 1.1	4.0 ± 2.1
--	0.03	-15.0 ± 1.4	1.5 ± 0.8
--	0.01	-12.0 ± 0.6	3.9 ± 0.1
DMN	10	93.4 ± 19.1 c	99.6 ± 0.4 c
--	1	93.4 ± 19.1 c	98.7 ± 0.8 c

a) NG (net nuclear grains) is the nuclear silver grain count minus the count in adjacent nuclear-size area of cytoplasm. 25 cells were counted for each of 3 slides per mouse. Values represent means +/- SE for 3 mice for each chemical concentration; b) the 10 mmol dose was cytotoxic as evidenced by sparse cultures, increased numbers of pyknotic nuclei, lowered nuclear grain counts and even greater lowering of cytoplasmic grain counts. The NG value for those cells remaining was -6.4 +/- 2.4; c) statistically significant difference compared to control group (unpaired t-test, p 0.05)

Conclusions:

No induction of DNA repair was observed at any of the tested chloroform concentrations.

Executive summary:

A test for chemically induced DNA repair was performed with chloroform in primary mouse hepatocyte cultures as described in Butterworth et al. (1987, Mutat. Res. 189, 113 -121) and mainly according to the OECD Guideline 482. Viable hepatocytes from each animal were incubated with 3 mL of Williams medium E containing either 0.01, 0.03, 0.1, 0.3, 1, 3 or 10 mmol of chloroform for 20 hr. Dimethylnitrosamine was used as the positive control. Net nuclear grains (NG) was calculated by counting the silver grains over the nucleus and subtracting the grain count from a nuclear-size area in the cytoplasm adjacent to the nucleus. NG were determined as population average. Any individual cell with a NG value of >= 5 was considered to be in repair. Statistical significance compared to controls was determined by the unpaired t-test for the equality of two means. A response was judged positive at p <= 0.05. No induction of DNA repair was observed at any chloroform concentration (Table 1). Concentrations of 10 mmol were toxic to the primary mouse hepatocyte cultures, as evidenced by pyknotic cells and loss of attachment of most of the cells. No UDS was evident in those few cells remaining at the 10 mmol dose of chloroform. The positive control, DMN, induced a strong DNA repair response, indicating that the cells were metabolically competent and responding to DNA damage. The current study indicated that chloroform is not directly genotoxic in hepatocytes of female mice in vitro, despite the fact that this is the target cell for tumor induction.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The majority of in vivo assays on the mutagenicity of chloroform produced negative results. Only in a few studies slightly positive results were observed. In order to clarify the uncertainties, a new mammalian erythrocyte micronucleus test was performed in Sprague-Dawley rats according to the current OECD and GLP guidelines (Whitwell 2009). This new in vivo assay did not show any genotoxic effect produced due to chloroform exposure.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 20C 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.

Key result

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.

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

Conclusions:

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.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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

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

Key result

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 genotoxic hepatocarcinogens tested (dimethylnitrosamine, 2-acetylaminofluorene...) produced strong positive response of >15.

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

Conclusions:

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).

Reference 3

Endpoint:

in vivo mammalian germ cell study: gene mutation

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

Principles of method if other than guideline:

In accordance with the lacI transgenic Big Blue(R) B6C3F1 mouse mutagenesis assay, Stratagene Cloning Systems (Provost et al 1993, Mutat Res 288, 133-149)

GLP compliance:

no

Type of assay:

other: lacl whole animal mutagenesis assay

Species:

mouse

Strain:

other: B6C3F1 lacI transgenic, Big Blue

Sex:

female

Details on test animals or test system and environmental conditions:

Animals were housed in chambers maintained on a 12 hours light/dark cycle at 22 +/- 2 °C and 50 +/- 10 % relative humidity. Chambers had a volume of 8 m3 and the air was pulled through the chambers at 225 L/min.

Route of administration:

inhalation

Vehicle:

none

Details on exposure:

Exposure atmospheres were generated by vaporisation technique. Nitrogen was used to pressurise the stainless steel storage vessels for chloroform and carry the vapours to the inlet of the exposure chambers. Generation and Characterization of Atmospheres: Exposures were conducted with mice housed individually in wire cages in 1-m3 chambers. Target airborne chloroform concentrations were 0, 10, 30, and 90 ppm. Mice were exposed by inhalation 6 hr/day 7 day/wk during the light cycle. The exposure atmospheres were generated by a vaporization technique. Chamber concentrations of chloroform were monitored using gas chromatography. Daily average analytical exposure concentrations were within 0.5% of the target for the 10, 30, and 90 ppm target concentrations, with a coefficient of variation of less than 4%. Chloroform exposure times were 10, 30, 90, or 180 days.

Duration of treatment / exposure:

6 hr/day for 10, 30, 90, or 180 days

Frequency of treatment:

daily

Post exposure period:

10 days after cessation of exposure

Remarks:

Doses / Concentrations:
0, 10, 30, or 90 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Ten

Control animals:

yes, concurrent no treatment

Positive control(s):

Dimethylnitrosamine

Tissues and cell types examined:

Treatment and Necropsy of Animals: The mutant frequency groups contained 10 animals per group for each dose and timepoint. Animals used for mutant frequency analysis were held without further treatment for 10 days after the last exposure day to allow for fixation of mutations. Induced cell proliferation was measured in the livers of separate, parallel groups (4-5 animals per group) of chloroform-treated lacl mice via inhalation, as above. Separate groups of mice (4 animals per group) were treated with 2, 4, or 8 mg/kg of DMN in water daily via oral gavage for 4 consecutive days. After sacrifice and exsanguination, the liver was removed and sections of the left and right median lobes were removed for analysis of histopathological changes and measurement of induced cell proliferation. The remaining liver was immediately frozen in liquid nitrogen and stored at -70°C.

Details of tissue and slide preparation:

Histopathology and Cell Proliferation: Sections of liver were evaluated for induced histopathological changes. In paraffin-embedded sections of liver from animals used to measure cell proliferation, at least 1,000 hepatocyte nuclei were scored to determine the hepatocyte BrdU labeling index. The labeling index (percentage of cells in S-phase) was calculated by dividing the number of hepatocyte nuclei that stained positive for BrdU incorporation by the total number of hepatocyte nuclei counted and the result expressed as a percentage.
Quantitation of Mutant Frequency: High-molecular weight genomic DNA was isolated from livers of treated animals intended for mutant frequency analysis. DNA isolated from livers of chloroform- and DMN-treated animals was packaged using Transpack packaging extract. Host bacteria were infected with packaged phage bacteria for approximately 20 min at 37°C. Blue mutant plaques were identified by examination of the plate against a red background.

Evaluation criteria:

The presence of histopathologic changes and the incidence of chloroform-induced cell proliferation in the livers of female mice treated with chloroform was checked. Then, the possible increase in the mutant frequencies (number of mutant plaques isolated per total plaques screened) in livers of treated animals relative to control mice was tested.

Statistics:

Student's t-test, significance when p <0.05

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Histopathological changes and cell-proliferation in livers of mice receiving exposure levels of 30 or 90 ppm

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Livers from DMN-treated mice exhibited a dose-related 2- to 5-fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable by lacI mutant frequency analysis, such as large deletions.

Conclusions:

Chloroform did not show mutagenic activity in this in vivo assay.

Executive summary:

The potential mutagenic activity of chloroform was evaluated in the B6C3F1 lacl transgenic mouse liver mutagenesis assay (Provost et al. 1993, Mutat Res 288, 133-149) to investigate chloroform-induced mutagenic events that might occur secondary to cytotoxicity. Animals were exposed 6hr/day by inhalation to 0, 10, 30 and 90 ppm of chloroform. Timepoints for determination of lacl mutant frequencies were 10, 30, 90 and 180 days of exposure. The repeated exposure to chloroform caused histopathological changes and cell proliferation in the livers of female B6C3F1 mice at levels of 30 and 90 ppm. No increase in lacl mutant frequency in the livers of treated animals were observed at any dose or timepoints with chloroform, indicating the absence of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable with a such test. Dimethylnitrosamine, a DNA-reactive mutagen and carcinogen, was used as positive control. Liver- from DMN-treated mice exhibited a dose-related 2-to 5-fold increase over control mutant frequency and remained at these levels for 10 through 180 days postexposure indicating the validity of the test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The weight of evidence indicates that chloroform is not a DNA-reactive mutagen.

Additional information

There is almost no indication for an interaction of chloroform or its metabolites formed in vivo with DNA in liver or kidney, which is supported by the lack of evidence in vivo for the induction of DNA strand breaks or repair and the absence of gene mutation induction in the mouse liver. A newly performed mammalian erythrocytes micronucleus test with Sprague Dawley rats receiving for five consecutive days doses of 120, 240 or 480 mg/kg via oral gavage concluded that chloroform did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of female and male Sprague-Dawley rats (Whitwell 2009). The analysis of the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes (MN PCE) in the negative vehicle control of the study were considered as valid. The clastogen positive control group exhibited statistically significantly increased numbers of MN PCE. Also the aneugenic positive control group produced significant upward shift in distribution of MN PCE. The test system therefore was considered as valid. Thus, this new study on the mutagenicity of chloroform gives evidence for the non-genotoxicity of chloroform. The formerly available information on clastogenic effects, which was used in the European Union risk assessment on chloroform, provided a less clear picture than the new mammalian erythrocytes micronucleus study. One study in Long Evans rats receiving chloroform via oral gavage or as intraperitoneal injection (Fujie et al. 1990) gave evidence for chromosomal aberrations in the bone marrow of rats. Another study provided mixed evidence by finding a significant increase in micronuclei but no chromosomal aberrations in mouse bone marrow (Shelby and Witt 1995). All other studies provided uncertain evidence or no evidence for a mutagenic effect of chloroform. However, the available data were not consistent in the responses with regard to the type of observation (micronucleus or chromosomal aberrations), species (rat or mouse) or target (most studies investigated bone marrow). The only positive study (Fujie et al. 1990) suggested that intraperitoneal administration caused greater sensitivity than oral administration (with bone marrow as the target), although significant responses were obtained with either route.

Justification for classification or non-classification

Based on all available data, particularly taking into account the negative results observed in the newly performed mammalian erythrocytes micronucleus test (Whitwell 2009), no classification of chloroform as a mutagen is required according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.