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 female and male B6C3F1 mice: implications for cancer risk assessment

Author:

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

Year:

1996

Bibliographic source:

Fund. Appl. Toxicol 30, 118-137

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

GLP compliance:

not specified

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 % purity and stabilised with 0.006 % amylenes obtained from Aldrich Chemical Co., Inc., Milwaukee, Wisconsin, USA was stored in 10-gallon stainless steel pressure vessels

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Seven-week old male and female B6C3F1 mice obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA; housed one per cage in 8-m3 stainless steel and glass inhalation chambers; separate chambers for each exposure concentration; acclimation for 2 weeks; chambers maintained on a 12-hours light/12-hours dark cycle, NIH-07 rodent chow (Ziegler Bros., Gardener; Pennsylvania), and deionised filtered tap water available ad libitum; temperature at 22.2 +/- 2 °C and 50 +/- 10 % relative humidity, continuous flow of HEPA and charcoal-filtered air at flow rates of 2000 litre/min

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

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 (see Table 1)

Duration of treatment / exposure:

see Table 2

Frequency of treatment:

6 hours on 5 days/week or 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.47 mg/m³ air

Remarks:

0.3 ppm (target concentration)

Doses / concentrations 2

Dose / conc.:

9.8 mg/m³ air

Remarks:

2 ppm (target concentration)

Doses / concentrations 3

Dose / conc.:

49 mg/m³ air

Remarks:

10 ppm (target concentration)

Doses / concentrations 4

Dose / conc.:

147 mg/m³ air

Remarks:

30 ppm (target concentration)

Doses / concentrations 5

Dose / conc.:

441 mg/m³ air

Remarks:

90 ppm (target concentration)

No. of animals per sex per dose:

ranging from 10 to 60, see Table 2

Control animals:

yes

Examinations

Sacrifice and pathology:

Mice 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 passages were examined as well

Statistics:

The Williams test was used to determine significant differences in organ weights, body weights and labelling index between treatment and control groups; the student's t-test was used to determine statistical differences in body weight 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:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

Animal health: No clinical signs of toxicity were noted at any time throughout the exposure study in females, and few of the male mice had rough hair coats and slight hair loss at the latter time points. No statistically significant differences between control and exposure groups were observed in body weight gain of females or males exposed for 13 weeks.
Organ weights: Relative liver weights were increased at each necropsy time point in female and male mice exposed to 90 ppm. No differences in relative kidney weights were observed between control and exposure groups at any time point.
Liver and kidney histopathology and cell proliferation: After 13 weeks of 7 days/week exposure, microscopic liver alterations (mild degenerative changes of slight centrilobular hepatocyte swelling and vacuolation) were present in 4/14 female mice exposed to 10 ppm and 10/15 mice exposed to 30 ppm. All female mice exposed to 90 ppm chloroform for 13 weeks at 5 days/week had mild changes of hepatocyte vacuolation and scattered enlarged nuclei. Increased cell proliferation was not observed at any time point with doses of 10 ppm or lower, but occurred at 3 and 6 weeks with doses of 30 ppm and at 13 weeks with doses of 90 ppm. Lesion scores for male mice exposed for 13 weeks at 7 days/week were increased above background at 30 and 90 ppm. Swollen centrilobular hepatocytes with pale eosinophilic cytoplasm, enlarged nuclei and centrilobular to midzonal hepatocyte vacuolation were observed. Significant increases in hepatocyte labelling index were found only in male mice exposed to 90 ppm.
Kidneys from female mice exposed to chloroform were not different histologically from controls at any time point. There was no statistically significant increase in the labelling index over controls in any of the treatment groups in the kidneys. Kidney lesions in male mice were confined to the epithelial cells of the proximal convoluted tubules of the cortex. Male mice exposed to 30 or 90 ppm had single or multiple foci of regeneration within the cortex. Mineralisation within the cortex was present in male mice exposed to 90 ppm. Enlarged nuclei were noted in PCT epithelial cells in male mice exposed to 10, 30 and 90 ppm. An increase in the labelling index was found in the epithelial cells of the PCT in the cortex and in the outer stripe of the outer medulla with 30 and 90 ppm, but not with 10 ppm or lower.
Nasal histopathology and cell proliferation: Nasal tissue had mild changes confined to the posterior ventral areas of the nose in and adjacent to the sites of attachment of the ethmoid turbinates to the lateral wall with doses of 10, 30 or 90 ppm. Nasal lesions were characterised by mild proliferative responses in the periosteum of the frontal and maxillary bones adjacent to the attachment of the osseous cores of the ethmoid turbinates. The response was characterised by a thickening of the normally delicate and regularly structured bone in this site through hypertrophy and hyperplasia of the periosteum and irregular formation of new, immature bone. The adjacent lamina propria exhibited variable loss of acini of Bowman's glands with mild vascular congestion and oedema.
Effects on other tissues: No microscopic changes were seen in the other tissues examined.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

9.8 mg/m³ air

Sex:

male

Basis for effect level:

other: based on renal and nasal changes at the next higher concentration of 49 mg/m3

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 3: Relative liver and kidney weight of female and male B6C3F1 mice exposed to chloroform vapours

	13 weeks of exposure
Concentration (ppm)	Liver	Kidney
Female mice
0	5.3 ± 0.3 b)	1.7 ± 0.1 b)
0.3	5.1 ± 0.3 b)	1.6 ± 0.2 c)
2	5.0 ± 0.2 b)	1.5 ± 0.1 b)
10	5.1 ± 0.2 b)	1.5 ± 0.2 b)
30	5.6 ± 0.7 b)	1.7 ± 0.1 b)
90	6.7 ± 0.3 b)*	1.6 ± 0.2 b)
Male mice
0	4.9 ± 0.3 b)	2.2 ± 0.1 b)
0.3	4.9 ± 0.3 b)	2.1 ± 0.2 b)
2	5.1 ± 0.3 b)	2.2 ± 0.1 b)
10	5.3 ± 0.2 b)	2.4 ± 0.2 b)
30	5.5 ± 0.1 b)*	1.9 ± 0.1 b)
90	6.3 ± 0.8 b)*	2.2 ± 0.2 b)

a) values are presented as organ weights as % of body weight; means +/- SD; b) n=5; * statistically different from controls (Williams test, p 0.05) Table 4: Hepatic lesion scores and incidence in female and male maic exposed to chloroform vapours a)

	Female mice			Male mice
Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	0.1 (1/15, 1.0)	0.1 (1/15, 1.0) b)	0.1 (1/15, 1.0) b)	0.3 (5/15, 1.0)	0.3 (5/15, 1.0) b)	0.3 (5/15, 1.0) b)
0.3	0.3 (1/15, 1.0)	c)	c)	0.4 (4/15, 1.5)	c)	c)
2	0.5 (5/14, 1.4)	c)	c)	0.3 (5/14, 1.0)	c)	c)
10	0.6 (4/14, 2.0)	0.2 (2/13, 1.0)	c)	0.3 (5/15, 1.0)	0.5 (4/13, 1.5)	c)
30	0.8 (10/15, 1.2)	c)	0.3 (1/8, 2.0)	1.2 (12/12, 1.2)	c)	0.6 (4/8, 1.3)
90	2.9 (15/15, 2.9)	1.8 (13/13, 1.8)	0.8 (2/8, 3.0)	1.9 (14/14, 1.9)	0.8 (10/12, 1.3)	0.8 (4/8, 1.5)

a) chloroform-induced liver histopathological changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of the lesions ranging from lipid vacuolation to degenerative changes and necrosis. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with liver lesions; b) control animals are the same for all the 13 -week studies; c) animals were not examined at these time points. Table 5) Hepatocyte labelling indices in female and male mice exposed to chloroform vapours

	Female mice			Male mice
Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	1.3 ± 1.0 e)	1.3 ± 1.0 g)	1.3 ± 1.0 g)	0.5 ± 0.4 e)	0.5 ± 0.4 g)	0.5 ± 0.4 g)
0.3	1.4 ± 1.0 e)	h)	h)	1.1 ± 0.8 e)	h)	h)
2	0.8 ± 0.3 f)	h)	h)	0.8 ± 0.6 f)	h)	h)
10	2.4 ± 1.5 f)	0.8 ± 0.3 c)	h)	1.0 ± 0.6 f)	1.5 ± 0.9 c)	h)
30	1.8 ± 1.4 f)	h)	1.0 ± 0.6 d)	0.9 ± 0.6 f)	h)	0.9 ± 0.7 d)
90	18.1 ± 8.5 e)*	7.1 ± 3.7 c)*	0.8 ± 0.6 c)	10.7 ± 4.9 e)*	3.1 ± 2.4 c)*	1.4 ± 0.9 c)

c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05) Table 6) Severity of nasal lesions in female mice exposed to chloroform vapours a)

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	0.1 ± 0.3 e)	0.1 ± 0.3 g)	0.1 ± 0.3 g)
0.3	0 e)	h)	h)
2	0.1 ± 0.3 e)	h)	h)
10	0.4 ± 0.5 f)	0.4 ± 0.5 d)	h)
30	0.4 ± 0.5 e)	h)	0 d)
90	0.1 ± 0.3 c)	0.3 ± 0.5 d)	0 d)

a) chloroform-induced nasal histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of connective tissue proliferation in the lamina propria. The severity of the nasal lesion for each group is expressed as the mean score +/- SD; c) n=8; d) n=7; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points Table 7: Nasal turbinate lamina propria labelling indices in female mice exposed to chloroform vapours a)

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	27 ± 9 e)	27 ± 9 g)	27 ± 9 g)
0.3	29 ± 27 e)	h)	h)
2	29 ± 17 d)	h)	h)
10	67 ± 33 d)	27 ± 18 b)	h)
30	43 ± 29 e)	h)	18 ± 8 b)
90	27 ± 6 b)	35 ± 8 e)	14 ± 5 e)***

a) all values are expressed as means +/- SD, b) n=5; d) n=7; e) n=4; g) control animals are the same for all the 13 -week duration studies; h) animals were not examined at these time points; *** statistically significant decreased compared to controls (Williams test, p 0.05) Table 8: Renal lesion scores and incidence in male mice exposed to chloroform vapours a)

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
0	0.0 (0/15, 0.0)	0.0 (0/15, 0.0) b)	0.0 (0/15, 0.0)
0.3	0.0 (0/15, 0.0)	c)	c)
2	0.0 (0/14, 0.0)	c)	c)
10	0.0 (0/15, 0.0)	0.1 (1/13, 1.0)	c)
30	0.9 (11/12, 1.0)	c)	1.0 (8/8, 1.0)
90	1.5 (14/14, 1.5)	1.6 (12/12, 1.6)	1.3 (8/8, 1.3)

a) chloroform-induced kidney histologic changes were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal; 2 = mild; 3 = moderate; 4 = severe; where 1 through 4 indicate increasing severity of nephropaty, which includes degenerative changes, foci of regeneration and mineralisation within the cortex. The top number is the mean lesion score for the entire group. The ratio in parentheses is that of the number of animals present with a kidney lesion relative to the total number of animals evaluated in that group. The second number in the parentheses is the mean lesion score for only that subset of animals with kidney lesions; b) control anaimals are the same for all the 13 -week studies; c) animals were not examined at these time points Table 9: Labelling indices in sections of the kidneys in male mice exposed to chloroform vapours a)

Concentration (ppm)	13 weeks, 7 days/week	13 weeks, 5 days/week	13 weeks, exposure stop after 6 weeks
Cortex
0	1.6 ± 0.6 f)	1.6 ± 0.6 g)	1.6 ± 0.6 g)
0.3	1.9 ± 0.8 f)	h)	h)
2	1.6 ± 0.6 e)	h)	h)
10	2.0 ± 0.9 f)	7.6 ± 3.0 c)*	h)
30	2.4 ± 1.2 f)*	h)	1.3 ± 0.4 c)
90	3.0 ± 0.9 e)*	5.7 ± 2.2 c)*	1.8 ± 0.3 c)
Outer stripe of outer medulla
0	0.8 ± 0.3 f)	0.8 ± 0.3 g)	0.8 ± 0.3 g)
0.3	1.1 ± 0.3 f)	h)	h)
2	0.8 ± 0.3 e)	h)	h)
10	0.8 ± 0.3 f)	1.6 ± 0.8 c)*	h)
30	0.6 ± 0.3 f)	h)	1.0 ± 0.5 c)
90	1.0 ± 0.4 e)	1.4 ± 0.6 c)*	0.9 ± 0.2 c)

a) values are expressed as means +/- SD; c) n=8; e) n=9; f) n=10; g) control animals are the same for all the 13 -week studies; h) animals were not examined at these time points; * statistically different from control (Williams test, p 0.05)

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of B6C3F1 mice to chloroform caused mild nasal lesions of the ethmoid region, liver cell proliferation and kidneys cell proliferation in female and male mice; the NOAEC was 2 ppm (9.8 mg/m3).

Executive summary:

A 90-day repeated dose inhalation toxicity study was carried out with chloroform using male and female B6C3F1 mice exposed to chloroform vapours at concentrations of 0, 0.3, 2, 10, 30 or 90 ppm (0, 1.47, 9.8, 49, 147 and 441 mg/m3) for 6 hours/day for 5 or 7 days per week. The liver of male and female animals was affected by exposure in a dose-dependent manner. The kidney of male mice was also affected by exposure. The exposure setting, i.e. groups having regular exposure interruptions for two days/week and groups receiving continuous exposure for 7 days a week and the duration of exposure, had an influence on the observed systemic effects. Increases in the relative liver weight in male and female mice were seen only at exposure to 90 ppm (441 mg/m3) chloroform. Liver lesions in male and female mice occurred at exposure concentrations of 30 (147 mg/m3) ppm or greater. The NOEC for regenerative cell proliferation in the liver of female mice was 10 ppm (49 mg/m3) and in the liver of male mice was 90 ppm (441 mg/m3). Kidneys of female mice were not different histologically from controls at any exposure concentration or time point. In contrast, lesions in the kidneys of male mice (enlarged nuclei in PCT epithelial cells and scattered areas of regenerating foci) were observed at exposure concentrations of 10 ppm (49 mg/m3). At 13 weeks, increased regenerative cell proliferation in the kidneys occurred at 30 ppm (147 mg/m3) when male mice were exposed continuously for 7 days a week and at 10 ppm (49 mg/m3) when male mice were exposed for 5 days a week. Local effects in the respiratory tract consisting of nasal lesions and induced cell proliferation essentially were confined to male and female mice exposed to concentrations of 10 ppm (49 mg/m3) or greater. These effects were observed only at the early time point after 4 days and were transient during the remainder of the study. In conclusion, male mice tended to be more susceptible to inhalation exposure to chloroform vapours than female mice. Male mice exhibited lesions of the kidney cells and increased regenerative cell proliferation in the kidneys at exposure concentration of 10 ppm (49 mg/m3), which was the LOAEC in the study; the NOAEC was set at 2 ppm (9.8 mg/m3).