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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline, well-conducted study, adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Evidence for the lack of base-change and small-deletion mutation induction by trichloroethylene in lacZ transgenic mice.
Author:
Douglas GR, Gingerich JD, Soper LM, Potvin M, Bjarnason S.
Year:
1999
Bibliographic source:
Environ Mol Mutagen; 34(2-3):190-4.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Testing of the ability to induce gene mutations and small deletions using the lacZ transgenic mouse model (MutaTMMouse)
GLP compliance:
not specified
Type of assay:
transgenic rodent mutagenicity assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): trichloroethylene (anhydrous)
- Source: Aldrich Chemical, Milwaukee, WI
- Analytical purity: 99+%

Test animals

Species:
mouse
Strain:
other: transgenic mouse strain (40.6)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Transgenic mouse strain (40.6). The transgene is based on a recombinant lgt10 vector containing the complete 3096 bp E. coli lacZ gene. Animals were maintained and treated under conditions approved by the Health Protection Branch Animal Care Committee.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 45-50
- Air flow: 500 l/min (10 chamber volumes/hr; HEPA and activated charcoal-filtered air)

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
none
Details on exposure:
The animals were transferred to 2.5-m3 inhalation chambers, into individual suspended stainless steel wire–mesh cages. The chambers were operated at an air flow of 500 l/min (about 10 chamber volumes/hr; HEPA and activated charcoal–filtered air), with an internal temperature of 19–23°C and relative humidity of 45–50%. Animals were exposed to trichloroethyle by inhalation for 6 hr per day for 12 days. The target concentrations selected were 0, 200, 1000, and 3000 ppm TCE. The atmospheres were produced by evaporating the substance through a glass evaporative system. Subsequently, the vapour was carried by an air stream into the chamber inlet and mixed with the incoming air. The chamber concentrations were monitored using an Amscor Xtra process gas chromatograph (Amscor, Angleton, TX) connected to the chambers through a multivalve system. Flow of the substance into the evaporator was adjusted to provide the required atmospheres in the chambers.
Duration of treatment / exposure:
12 days
Frequency of treatment:
6 hr/day
Post exposure period:
2 days or 48 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 1000 and 3000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
203±10, 1153±361, and 3141±378 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5-10
Control animals:
yes
Positive control(s):
None

Examinations

Tissues and cell types examined:
High molecular weight DNA was isolated from: lung, liver, bone marrow, spleen, kidney and testicular germ cells
Details of tissue and slide preparation:
DNA isolation
Following sacrifice by cervical dislocation 14 or 60 days after exposure, tissues were removed, placed in storage vials, frozen in liquid nitrogen, and maintained at 280°C until used. Bone marrow was flushed from femurs with PBS before freezing. High molecular weight DNA was isolated from defrosted tissues as follows:

Bone marrow. Tissue was resuspended in lysis buffer (10 mM Tris, pH 7.6; 150 mM NaCl; 10 mM EDTA), digested overnight in 1.0% SDS and 1 mg/ml proteinase K (GIBCO/BRL, Gaithersburg, MD) in lysis buffer at 37–40°C, then extracted once with phenol/chloroform and once with chloroform. The DNA was precipitated with ethanol.

Kidney, spleen. Thawed tissues were minced in PBS, and then processed as per bone marrow.

Liver. Prior to digestion, nuclei were isolated by homogenizing the tissue in TMST (50 mM Tris, pH 7.6; 3 mM magnesium acetate; 250 mM sucrose; 0.2% Triton X-100). The nuclei were centrifuged at 1200 g for 6 min, washed twice in TMST, then processed as per bone marrow.

Lung. Lung tissue from one lung per animal was minced with scissors and transferred to a centrifuge tube containing 2 ml of PBS. A vacuum was applied to remove air from the tissue. The tissue samples were centrifuged, washed again in PBS, suspended in lysis buffer, and processed as per bone marrow.

Testicular germ cells. The membrane surrounding the thawed testis was removed. Cells were extruded into PBS from the seminiferous tubules using a small roller on a ground glass plate. The cells collected were centrifuged then processed as per bone marrow.
Evaluation criteria:
not specified
Statistics:
not specified

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
TCE, administered by inhalation for 6 hr/day for 12 days, did not induce (base-change or small-deletions) gene mutations at any of the doses tested in male and female lung, liver, bone marrow, spleen, and kidney, or in male testicular germ cells when animals were sampled 60 days following exposure. The 60-day sampling time was selected because it is believed to accommodate any differences in manifestation times among the tissues selected.
In addition, gene mutations were not observed in the lungs at 14 days after the end of exposure. Despite the likelihood that the sampling times used were adequate to capture detectable mutations, the possibility may exist that mutations were fixed after the 14-day sampling time, and were missed due to tissue turnover, or cell death, prior to sampling at 60 days.

Applicant's summary and conclusion