Chloroform

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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:

Toxicity and cell proliferation in the liver, kidneys and nasal passages of female F-344 rats induced by chloroform administered by gavage

Author:

Larson JL, Wolf DC, Méry S, Morgan KT, Butterworth BE

Year:

1995

Bibliographic source:

Fd Chem. Toxicol. 33(6), 443-456

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

animals were exposed 5 d/wk for 3 wks

GLP compliance:

no

Limit test:

no

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:

chloroform, > 99.5 % and stabilised with 0.006 % amylenes, purchased from Fluka Chemika (Buchs, Switzerland)

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina, USA)
- Age at study initiation: 9 weeks
- Weight at study initiation: 139.5 +/- 5 g
- Fasting period before study: no data
- Housing: rats were housed two or three per cage in polystyrene shoe-box cages with cellulose bedding (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, USA), and filter top lids
- Diet (e.g. ad libitum): NIH-07 rodent chow (Ziegler Bros, Gardener, Pennsylvania, USA) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Rats received chloroform dissolved in corn oil at doses of 0, 34, 100, 200 and 400 mg/kg/day in a constant dosing volume of 2 mL/kg administered by oral gavage. Each dose concentration of chloroform was prepared fresh on the morning that it was dispensed. All doses were administered between 8.00 and 10.00 hr to minimise diurnal variation.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 consecutive days or 3 weeks

Frequency of treatment:

Daily in the 4-day study and one administration per day on five days per week in the 3-week study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

34 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 rats per dose group

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

no data, but regular observations were performed during the testing period

Sacrifice and pathology:

At autopsy, rats were anaesthetised with sodium pentobarbital and the kidneys perfused in situ. Following a 3-min perfusion, the entire livers and kidneys were removed. Livers were weighed, examined macroscopically, and longitudinal 3-4 mm mid-sections of the left, median and right anterior hepatic lobes taken. The right and left kidneys were examined grossly and a 3-4 mm mid-saggital section of the left kidney and a 3-4 mm midtransverse section of the right kidney were taken. A 3-4 mm section of the duodenum was also included to confirm the systemic distribution and nuclear incorporation of bromodeoxyuridine (BrdU). Nasal cavities were flushed with neutral buffered 10 % formalin through the trachea; excess soft tissue was removed from the head, the lower jaw was removed, and the head was subsequently immersed in fresh fixative for no less than 48 hours and then decalcified in 5 % formic acid with ion exchange resin. Uniform blocks were cut at six levels of the nose to include all major epithelial types and blocks processed to paraffin. Tissue sections were cut at 4-5 micrometre thick. Serial sections were stained with haematoxylin and eosin or left unstained for BrdU immunohistochemistry.

Other examinations:

The labelling index (percentage of nuclei in S-phase) was evaluated in rats by bromodeoxyuridine (BrdU) immunohistochemistry. On the Monday afternoon preceding a Friday autopsy, osmotic pumps containing 20 mg/mL BrdU were aseptically implanted sc over the thoracumbular area using isofluorane anaesthesia. Autopsies were conducted on Friday mornings; consequently, rats received BrdU for about 3.5 days. BrdU-labelled tissues were mounted on "ProbOn Plus" slides (Fisher Scientific, Pittsburg, Pennsylvania, USA) to ensure adhesion during processing. The immunohistochemical detection of BrdU-labelled cells was done as described by Elridge et al. (1990, Carcinogenesis 11, 2245-2251). Cells that had incorporated BrdU were identified by the red pigment within the nuclei. Computer-generated random fields were used for scoring BrdU-labelled nuclei in the liver and kidney by light microscopy. In the liver tissue sections, at least 1000 hepatocellular nuclei in the left lobe were counted. For kidney tissue sections, at least 1000 proximal tubular cells in the cortical region were scored, as well as 1000 epithelial cells in the outer stripe of the medulla, 1000 epithelial cells in the inner stripe of the outer medulla and 1000 epithelial cells in the inner medulla. The labelling index, representing the percentage of nuclei in S-phase, was calculated by dividing the number of labelled nuclei by the total number of nuclei counted (expressed in %). For the nasal passages, quantitative studies of cell replication were confined to the base of the dorsal scroll of the first endoturbinate. These studies used the unit length labelling index method as modified by Méry et al. (1994, Toxicology and Applied Pharmacology 125, 214-227) for investigation of nasal lesions.

Statistics:

The Williams test was used to determine significant differences in liver and body weights and labelling index between treatment and control means. The Williams test is designed to establish the lowest dose that produces a response that is significantly elevated over the control in a dose-response study.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mild degenerative centrilobular changes and dose-dependent increases in the hepatocyte labelling index (LI ) were observed after administration of 100 mg or more chloroform/kg/day. Rats given 200 or 400 mg/kg/day for 4 days or 3 wk had degeneration and necrosis of the proximal tubules of the renal cortex. Regenerating epithelium lining proximal tubules was seen histologically and as an increase in LI. Dose-dependent increases in LI were observed in the kidneys at doses of 100 mg or more chloroform/kg/day at both 4 days and 3 wk. Two distinct treatment-induced responses were observed in specific regions of the olfactory mucosa lining the ethmoid region of the nose. A peripheral lesion was seen at all doses used and included new bone formation, periosteal hypercellularity and increased cell replication. A central lesion was seen at doses of 100 mg or more chloroform/kg/day and was characterized by degeneration of the olfactory epithelium and superficial Bowman's glands. These observations define the dose-response relationships for the liver, kidneys and nasal passages as target organs for chloroform administered by gavage in the female F-344 rat.

Effect levels

Dose descriptor:

NOAEL

Effect level:

34 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: changes in liver and kidneys at the next higher level of 100 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Chloroform-induced proliferation in the liver of female F-344 rats

Dose (mg/kg/day)	Labelling index in hepatocytes after:
	4 days	3 weeks
0	1.6 ± 1.1	0.6 ± 0.5
34	1.0 ± 0.6	0.8 ± 0.4
100	6.0 ± 2.5 *	2.7 ± 1.5
200	11.7 ± 3.1 *	14.0 ± 9.0 *
400	14.0 ± 5.7 *	11.8 ± 15.9 *

Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)

Table 2: Chloroform-induced cell proliferation in the kidneys of female F-344 rats

Duration of administration	Dose (mg/kg/day)	Labelling index in:
		Cortex	Outer stripe, outer medulla	Inner stripe, outer medulla	Inner medulla
4 days	0	2.1 ± 0.6	2.9 ± 0.7	1.4 ± 0.7	0.4 ± 0.4
	34	2.4 ± 0.8	2.1 ± 0.9	2.0 ± 1.3	0.8 ± 0.7
	100	3.2 ± 0.7	1.7 ± 0.5	1.0 ± 0.4	0.6 ± 0.3
	200	17.7 ± 12.2 *	9.1 ± 16.0	6.6 ± 12.1	2.3 ± 4.3
	400	41.0 ± 5.3 *	31.1 ± 2.2 *	27.3 ± 4.1 *	3.2 ± 1.8 *
3 weeks	0	1.3 ± 1.0	1.2 ± 0.8	1.2 ± 0.7	0.4 ± 0.3
	34	1.5 ± 0.3	1.0 ± 0.5	1.1 ± 0.6	0.2 ± 0.3
	100	22.4 ± 20.9 *	4.9 ± 6.8	3.3 ± 3.9	0.6 ± 0.6
	200	33.8 ± 20.9 *	10.4 ± 12.0 *	6.1 ± 7.1	5.1 ± 7.5
	400	13.5 ± 6.9 *	5.7 ± 2.4 *	2.0 ± 1.4	0.5 ± 0.6

Values are means ± SD (n = 5 rats); Asterisks indicate significant differences from the control (p < 0.05; William’s test)

Table 3: Severity of chloroform-induced nasal lesions (mean subjective score) in femal F-344 rats

Dose (mg/kg/day)	Peripheral olfactory mucosal lesion a)		Central olfactory mucosal lesion b)
	4 days	3 weeks	4 days	3 weeks
0	0	0	0	0
34	1.2	0.4	0.2	0
100	1.8	1.7	0.4	0.8
200	1.6	2.0	0.8	1.2
400	1.8	3.0	2.8	1.6

a) Periosteal hyperplasia, new bone formation, and loss of adjacent Bowman’s glands. Grading system for the turbinate bones: 0 = normal, 1 = uneven boundary of nasal bone, 2 = uneven boundary of nasal bone with slight new bone formation, 3 = moderate new bone formation, 4 = severe bone enlargement. Standard deviations are not presented owing to the subjective nature of the data and the consistent intragroup scores.

b) Degeneration of olfactory epithelium and superficial Bowman’s glands. Scoring system used for degeneration of the olfactory epithelium of the dorsal medial region of the nose: 0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.

Table 4: Chloroform-induced cell proliferation in the nasal turbinates of female F-344 rats.

Dose (mg/kg/day)	ULLI a)
	4 days	3 weeks
0	15 ± 4	16 ± 3
34	145 ± 97 *	24 ± 9
100	306 ± 48 *	61 ± 10 *
200	321 ± 19 *	63 ± 5 *
400	377 ± 121 *	63 ± 17 *

a) Unit length labelling index of cells in the lamina propria of the proximal portion of the dorsal scroll of the first endoturbinate expressed as labelled nuclei per 0.25 mm bone. Values are means ± SD. Asterisks indicate significant differences from the control (p < 0.05, William’s test).

Applicant's summary and conclusion

Conclusions:

The LOAEL for oral exposure to chloroform dissolved in corn oil administered by gavage in a subacute study using female F-344 rats was 34 mg/kg body weight/day after exposure for 4 consecutive days; the NOAEL was 34 mg/kg body weight/day after exposure for three weeks. The LOAEL was based on observations of lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages.

Executive summary:

A study on the sub-acute toxicity of chloroform was carried out using female F-344 rats exposed by oral gavage to graded doses of chloroform dissolved in corn oil for 3 consecutive weeks on 5 days per week. The study was comparable to a guideline study according to method B.7 of the European Commission with acceptable restrictions. Liver, kidneys and nasal passages were identified as the target organs for chloroform administered by oral gavage. Mild degenerative centrilobular changes and dose-dependent increase in the hepatocyte labelling index (% of cells in S-phase), dose-dependent increase in labelling index in the kidneys, lesions and cell proliferation in the olfactory epithelium and changes in the nasal passages were observed at 100 mg/kg body weight/day of chloroform after 3 weeks of exposure. The NOAEL for systemic effects was 34 mg/kg body weight/day based on the finding of lesions and increased cell replication in the liver and kidneys at higher dose levels observed after repeated oral doses administered for 3 weeks. Local effects in the nasal passage were seen after 4 days of exposure at a dose of 34 mg/kg/day.