Chloroform

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was carried out according to principles similar to those laid down in the relevant guidelines for testing of acute inhalation toxicity (OECD No. 403) with acceptable restrictions. The scoring of the nasal histologic changes was done according to the scoring system of skin irritation effects (OECD No. 404). The experimental settings and the results are presented in detail. The results are considered to be reliable with 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:

other: OECD Guideline No. 403

Principles of method if other than guideline:

Scoring system for nasal histologic changes similar to that of skin irritation effects (OECD Guideline No. 404)

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

respiratory irritation

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, stabilised with 0.006 % amylenes, obtained from Aldrich Chemical Co., Inc., Milwauke, Michigan, USA, stored in 10-gallon stainless steel vessels

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Obtained from Charles River Breeding Laboratories, Inc., Raleigh, North Carolina, USA,
- Age at study initiation: 9 weeks
- Weight at study initiation: no data
- Housing: housed one per cage in 8 m3 stainless steel and glass inhalation chambers
- Diet (e.g. ad libitum): NIH-07 rodent chow (Ziegler Bros., Gardener, Pennsylvania, USA) ad libitum
- Water (e.g. ad libitum): deionised, filtered tap water ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 +/- 2
- Humidity (%): 50 +/- 10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

The target exposure concentrations of chloroform were 0, 0.3, 2, 10, 30 and 90 ppm. The exposure atmospheres were generated by a vaporisation technique. Chloroform was stored in 10-gallon stainless steel pressure vessels. A nonpressurised system was used in the 30- and 90-ppm chambers. Nitrogen, metered by a mass flow controller, was admitted into the vessel through a dip tube with its opening located below the surface of the chloroform. For the 0.3-, 2-, and 10 -ppm chambers, nitrogen was used to pressurise the vessel to 5 psi and carry the chloroform into the chambers through a mass flow controller. Chamber concentrations of chloroform were monitored using a Miran 1A infrared gas analyser. Certified gas standards were used to check the calibration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations were monitored using a Miran 1A infrared gas analyzer.

Duration of treatment / exposure:

6 hours per day on 4 consecutive days
6 hours per day on 7 days per week for 3 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.3, 2, 10, 30, 90 ppm

No. of animals per sex per dose:

5 female mice per exposure level of 0, 0.3, 2, 10, 30 or 90 ppm in the 4-days study
8 female and 8 male mice per exposure level of 0, 0.3, 2 or 10 ppm and 5 female and 5 male mice per exposure level of 30 or 90 ppm

Details on study design:

Animals were exposed to chloroform during 6 hours per day on 4 consecutive days. Mice were administered Bromodeoxyuridine (BrdU) via subcutaneously implanted osmotic pumps approximately 3.5 days prior to necropsy to label cells in S-phase. A complete soft-tissue screen was collected for mice exposed for 3 weeks.

Results and discussion

Details on results:

Regular inhalation of chloroform vapours over a short duration in mice induced transient, locally increased cell replication, transient hypercellularity of the lamina propria and other mild nasal lesions of the lateral ethmoid region.

Any other information on results incl. tables

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. These responses were confined to animals exposed to 10, 30, or 90 ppm for 4 days. This response was transient, with little or no difference from controls in the other time or concentration groups. BrdU-stained sections revealed clear increases in the number of S-phase nuclei in the lamina propria with a distribution pattern identical to that described above for microscopical changes. The base of the first endoturbinate was selected for quantitative studies because this region exhibited histologic changes. There was a concentration-related induced cell proliferation response to chloroform in mice exposed to 10, 30, or 90 ppm chloroform. The response was transient and essentially confined to animals exposed for 4 days. Female mouse nasal lesions were characterised by mild proliferative responses in the periosteum of the frontal and maxillary bones adjacent to the atttachment of the osseous cores of the ethmoid turbinates. This 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 but minimal formation of new, immature bone. The adjacent lamina propria exhibited variable loss of acini of Bowman's glands with mild vascular congestion and oedema. The distribution of these lesions was consistent in all affected mice.

Table 1: Severity of nasal lesions and nasal turbinate lamina propria labelling indices in female B6C3F1 mice exposed ot chloroform vapours.

Concentration (ppm or mg/m3)	4 days		3 weeks, 7 days per week		6 weeks, 7 days per week		13 weeks, 7 days per week
0	0 a)	15 ± 8 a)	0.3 ± 0.5 b)	21 ± 5 b)	0 b)	24 ± 6 c)	0.1 ± 0.3 d)	27 ± 9 f)
0.3 or 1.47	0 a)	9 ± 3 f)	0.2 ± 0.4 b)	23 ± 6 g)	0.4 ± 0.5 b)	19 ± 3 f)	0 d)	29 ± 27 f)
2 or 9.8	0.5 ± 0.5 a)	16 ± 5 a)	0.1 ± 0.4 b)	59 ± 15 c)***	0.5 ± 0.5 b)	27 ± 10 f)	0.1 ± 0.3 d)	29 ± 17 c)
10 or 49	1.6 ± 0.5 a)	164 ± 49 a)***	0.8 ± 0.5 b)	42 ± 13 c)***	0.3 ± 0.5 c)	33 ± 12 c)	0.4 ± 0.5 e)	67 ± 33 c)
30 or 157	1.8 ± 1. 0 a)	281 ± 151 g)***	0.2 ± 0.4 a)	23 ± 4 g)*	0.5 ± 0.5 a)	27 ± 8 f)	0.4 ± 0.5 d)	43 ± 29 f)
90 or 441	2.4 ± 0.5 a)	397 ± 27 f)***	0.2 ± 0.4 a)	26 ± 9 a)*	0 a)	28 ± 12 a)*	0.1 ± 0.3 b)	27 ± 6 a)

a) n=5; b) n=8), c) n=7; d) n=9; e) n=10; f) n=4; g) n=3 * statistically significant increased compared to controls (Williams test, p 0.05); *** statistically significant decrease compared to controls (Williams test, p 0.05)

Applicant's summary and conclusion

Conclusions:

Regular inhalation of chloroform vapours over a short duration in mice induced transient, locally increased cell replication, transient hypercellularity of the lamina propria and other mild nasal lesions of the lateral ethmoid region. The NOAEC in the present study was 2 ppm (9.8 mg/m3).

Executive summary:

The potential of chloroform to induce acute respiratory irritation was tested in female B6C3F1 mice exposed to chloroform vapours during 6 hours per day on 4 consecutive days or during 6 hours per day on 7 days per week for 3 weeks. The principles of the study were similar to those laid down in the OECD Guidelines No. 403 and No. 404. Nasal lesions and significant proliferation in mice occurred at doses of 10 ppm (49 mg/m3) and above, but only at the early time point. The occurring lesions were transient and no longer discernible after three weeks of exposure. Lesions were observed in regions of the nose lined by olfactory mucosa, specifically the lateral and ventral regions of the ethmoid turbinates and adjacent nasal wall, while they spared the dorsal portion of the nasal septum. The NOAEC in the present study was 2 ppm (9.8 mg/m3).