(2H)chloroform

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

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:

A 90-day chloroform inhalation study in F-344 rats: profile of toxicity and relevance to cancer studies

Author:

Templin MV, Larson JL, Butterworth BE, Jamison KC, Leininger JR, Méry S, Morgan KT, Wong BA, Wolf DC

Year:

1996

Bibliographic source:

Fundamental and Applied Toxicology 32, 109-125

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

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:

chloroform

Test material form:

liquid

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

7-week old male and female F-344 rats (CDF(F-344)/CrBR) were obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; rats were housed one per cage in 8 m3 stainless steel and glass inhalation chambers; animals were randomised by weight and assigned to control or treatment groups with separate chambers used for each exposure concentration; chambers were maintained on a 12-hr light-dark cycle; temperature was at 22.2 +/- 2 °C, humidity was 50 +/- 10 %, chambers were operated with a continuous flow of HEPA- and charcoal filtered air at 2000 litre/minute; rats were acclimated in the chambers for 2 weeks and were 9-weeks old at the start of exposure; NIH-07 rodent chow (Ziegler Bros., Gardener, Pennsylvania, USA) and deionised filtered tap water were available ad libitum; food was changed daily; animals were weighed before the beginning of the test and then biweekly

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Exposure atmosphere was generated by a vaporisation technique; Nitrogen, metered by a mass flow controller, was admitted into the chloroform storage vessel through a dip tube; the chloroform-containing nitrogen gas flowed into the supply air duct for the exposure chamber; in the 2 and 10 ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chamber through a mass flow controller

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser; average analytical exposure concentrations were always within 4.5 % of the target with standard deviations of no more than 2.7 % from the mean

Duration of treatment / exposure:

Daily exposures were conducted for 6 hours, for duration of treatment see Table 1

Frequency of treatment:

see Table 1

Doses / concentrations

Remarks:

Doses / Concentrations:0, 2, 10, 30, 90, 300 ppmBasis:nominal conc.

No. of animals per sex per dose:

see Table 1

Control animals:

yes

Details on study design:

see Table 1

Examinations

Sacrifice and pathology:

Rats in unlabelled group were weighed and killed subsequently; livers and kidneys were removed, weighed and examined macroscopically, then slides were prepared from the tissues; in rats exposed for 3 or 13 weeks, a complete tissue screen was collected including adrenals, brain, cecum, cervix, colon, duodenum, ear canal, esophagus, eye with haderian gland, femoral-tibial joint, heart, ileum, jejunum, kidneys, larynx, liver, lungs, mesenteric lymph nodes, ovaries, pancreas, parathyroid gland, ribs, prostate, salivary gland, skin with mammary gland, sciatic nerve, seminal vesicles, spinal cord, sternum, stomach, spleen, testes, thigh muscle, thymus, thyroid, trachea, urinary bladder, uterus, vagina and vertebrae.

Other examinations:

Nasal cavities were examined as well

Statistics:

The Williams test was used to determine significant differences in organ weights, bodyweights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in bodyweight and organ weights and labelling index between the exposure groups of 7 and 5 days/week and between exposure groups of 13-week continuous chloroform exposure and 6-week stop.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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 examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Signs of mild dehydration were observed in some rats at early time points, slight hair loss, discharge from the eyes and anogenital staining were noted at later time points. These observations were confined primarily to the high dose groups. Dose-dependent decreases in body weight gain were observed in chloroform-exposed rats at all time points. This effect was most pronounced in animals exposed to 90 or 300 ppm. Significant increases in kidney and liver weights were observed in animals exposed to 90 or 300 ppm, with effects more pronounced in female rats. Details of the renal, liver and nasal histopathology are given in Table 2, Table 3 and Table 4.

Effect levels

Dose descriptor:

LOAEC

Effect level:

2 ppm

Sex:

male/female

Basis for effect level:

other: enhanced bone growth and hypercellularity in the lamina propria of the ethmoid turbinates

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2: Kidney lesion scores and incidence in male and female F-344 rats exposed to chloroform vapours

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
Male rats
0	0.6 (8/14)	0.6 (8/14) b)	0.6 (8/14) b)
2	0.8 (10/15)	c)	c)
10	0.5 (7/15)	c)	c)
30	0.6 (9/14)	0.1 (2/15)	c)
90	1.2 (14/15)	0.6 (6/13)	1.1 (8/8)
300	1.4 (14/14)	2.8 (13/13)	1.4 (8/8)
Female rats
0	0.4 (6/14)	0.4 (6/14) b)	0.4 (6/14) b)
2	0.7 (10/15)	c)	c)
10	0.7 (10/15)	c)	c)
30	0.8 (12/15)	1.8 (13/13)	c)
90	0.7 (10/15)	0.4 (5/13)	0.9 (7/8)
300	1.1 (14/14)	1.4 (13/13)	0.8 (6/8)

a) chloroform-induced kidney histopathological changes were scored qualitatively as follows: 0 = within normal limits; 1 0 minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from vacuolation of proximal cell tubule (PCT) epithelium, enlarged PCT nuclei, pyknotic PCT nuclei, to individual tubule cell necrosis. The first number in each box is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points Table 3: Hepatic lesion scores and incidence in male and female F-344 rats exposed to chloroform vapours

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
Male rats
0	0.1 (1/15)	0.1 (1/15) b)	0.1 (1/15) b)
2	0.2 (3/15)	c)	c)
10	0.0 (0/15)	c)	c)
30	0.1 (2/15)	0.0 (0/13)	c)
90	1.0 (14/15)	0.3 (4/13)	0.0 (0/8)
300	3.9 (15/15)	2.4 (13/13)	0.0 (0/8)
Female rats
0	0.1 (1/15)	0.1 (1/15) b)	0.1 (1/15) b)
2	0.1 (1/15)	c)	c)
10	0.0 (0/14)	c)	c)
30	0.0 (0/15)	0.0 (0/13)	c)
90	0.8 (12/15)	0.3 (4/13)	0.1 (1/8)
300	3.0 (15/15)	2.0 (13/13)	0.0 (0/8)

a) chloroform-induced liver histopathological changes were scored qualitatively as follows: 0 = within normal limits; 1 0 minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from hepatocyte vacuolation, degenerative changes in hepatocytes to hepatocyte necrosis. The first number in each box is the mean lesion score for the entire group. The ratio in the parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points Table 4: Severity of nasal lesions in male F-344 rats exposed to chloroform vapours (effects in female rats were similar but not reported in the original publication).

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	0.0 (0/10)	0.0 (0/10) b)	0.0 (0/10) b)
2	1.1 (10/10)	c)	c)
10	2.0 (10/10)	c)	c)
30	2.0 (10/10)	1.8 (8/8)	c)
90	2.5 (10/10)	2.0 (8/8)	2.1 (5/8)
300	2.9 (10/10)	3.0 (8/8)	2.9 (8/8)

a) chloroform-induced histopathological changes in the ethmoid region of the nasal passage were scored qualitatively as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions. Severity scores were assigned for lesions ranging from oedemal and loss of Bowman's glands, olfactory metaplasia, basal lamina mineralisation to generalised atrophy of the ethmoid turbinates. The first number in each box is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a lesion score of 1 or greater relative to the total number of animals evaluated in the group; b) control animals are the same for all the 13 -week studies; c) animals were not exposed at these time points

Applicant's summary and conclusion

Conclusions:

Nasal effects were observed at all inhalation doses tested in the study starting from 2 ppm. The experimental NOAEL value for changes within the proximal tubules of the kidney cortex in female rats was found to be 10 ppm.

Executive summary:

A 90-day subchronic toxicity study was carried out in male and female F-344 rats according to principles similar to those of the OECD Guideline for the Testing of Chemicals No. 413 (with acceptable restrictions). Different groups of rats were exposed by inhalation to chloroform vapours at concentrations of 0, 2, 10, 30, 90, 300 ppm for 6 hours per day for 5 or 7 days per week for 13 weeks.

Systemic effects were seen in the kidneys and the liver of rats. At 13 weeks of exposure, scattered vacuolation of the PCT and enlarged epithelial cell nuclei were observed in male rats exposed to 90 ppm. Necrosis of individual tubule cells was found in male rats exposed to 300 ppm. Increased regenerative cell proliferation in the kidneys of male rats was seen with exposure concentrations of 30 ppm or greater. In female rats exposed for 13 weeks, significant lesions in the kidneys were observed at 300 ppm levels, however, histologic changes such as vacuolation in the cortex were seen at exposure levels of 30 ppm and greater. As in the male rats, increased regenerative cell proliferation was observed in the kidneys of female rats at exposure to 30 ppm or greater. Thus, the NOEC for adverse effects in the kidneys was 10 ppm for male and female rats, respectively.

At 13 weeks of exposure, hepatocyte alterations were found in male and female rats exposed to 90 or 300 ppm. An increase in regenerative cell proliferation in the liver of male and female rats was observed only at exposure to 300 ppm.

In conclusion, repeated inhalation exposure to chloroform vapours for 13 weeks resulted in local and systemic effects in male and female F-344 rats. The NOEC for increased regenerative cell proliferation and lesions in the kidneys of male and female rats was 10 ppm.