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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Version / remarks:
The assay was performed according to the current recommendations of the United Kingdom Environmental Mutagen Society (UKEMS) (Richold et al, 1990; Kennelly et al, 1993) and the most recent draft guidelines of the Organisation
for Economic Co-operation and Development (OECD).
GLP compliance:
no
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
Bromoform
EC Number:
200-854-6
EC Name:
Bromoform
Cas Number:
75-25-2
Molecular formula:
CHBr3
IUPAC Name:
bromoform
Specific details on test material used for the study:
Bromoform (CAS 75-25-2, >99% pure) was obtained from Aldrich Chemical Co., Gillingham, UK.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Pathogen-free Hsd/Ola outbred albino Sprague-Dawley male rats.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: pathogen-free Hsd/Ola outbred albino Sprague-Dawley male
rats were obtained from Harlan Olac UK Ltd, Bicester, UK.
- Age at study initiation: approx. 35 days old on dispatch from the supplier
- Weight at study initiation: 150-159g on dispatch from the supplier.
- Assigned to test groups randomly: Not stated
- Fasting period before study: Not stated
- Housing: Animals were group-housed in polystyrene disposable cages containing certified contaminant free woodchip bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4- 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 55%
- Air changes (per hr): 20 changes of air per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: To: Not stated

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
aqueous 1% w/v methylcellulose
Duration of treatment / exposure:
Single dose (time = 0) with sampling at 2 and 14 hours.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Remarks:
Vehicle Control
Dose / conc.:
324 mg/kg bw (total dose)
Dose / conc.:
1 080 mg/kg bw (total dose)
No. of animals per sex per dose:
4 animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
dimethylnitrosamine and 2-acetylaminofluorene

Examinations

Statistics:
Results were subjected to statistical analysis using classical one-way analysis of variance followed by a Student's t-test (Snedecor and Cochran, 1967).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs including lethargy, unsteady gait and piloerection were seen within 30 min of administration at the high doses of test susbtance confirming absorption of the compound.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Clinical signs including lethargy, unsteady gait and piloerection were seen within 30 min of administration of high levels of bromoform during preliminary testing confirming absorption of the compounds following oral administration. These signs gradually moderated over a period of up to 4 days (depending on the compound and dosage).


 


In the main test, a statistically significant increase in net nuclear grain count compared with the concurrent control was obtained at the 14 h sampling time for the high dose group of bromodichloromethane. Since this increase was small and was not accompanied by any increase in the gross nuclear grain count and since the net nuclear grain count was well within the laboratory historical control range (group means ranged from -4.3 to -0.7 net nuclear grains for 124 experiments, mean animal net grain count was -2.3 for 446 animals), it is not considered to be indicative of an increase in unscheduled DNA synthesis. No other significant increases in grain count were obtained for bromodichloromethane at either dose level or sampling time.


 


Bromoform did not caused any significant increases in the gross or net nuclear grain counts at either sampling time.

Applicant's summary and conclusion

Conclusions:
Bromoform did not show any DNA damaging activity in the rat liver UDS assay and is considered negative for genotoxicity in this assay.
Executive summary:

Introduction


The in vivo genotoxicity of Bromoform was investigated in the Rat Liver Unscheduled DNA Synthesis (UDS) test using a protocol performed according to the recommendations of the United Kingdom Environmental Mutagen Society (UKEMS) (Richold et ai, 1990; Kennelly et ai, 1993) and the most recent draft guidelines of the Organisation for Economic Co-operation and Development (OECD) available at the time of testing.


 


Method


The maximum tolerated dose (MTD) for bromoform was determined over a 4-day observation period. The estimated MTD for bromoform in the rat was 1080 mg/kg/bw.  This dose level was therefore selected as an appropriate maxima for use in the main test.


Groups of male rats were treated orally by gastric intubation using a standard dose volume of 10 ml/kg bodyweight with a single dose of the vehicle control (aqueous 1% w/v methylcellulose) or the test substance at 324 mg/kg/bw or 1080 mg/kg/bw.


 


Animals were killed by exposure to a gradually increasing atmospheric concentration of carbon dioxide. Hepatocytes were isolated and cultured from four animals in each group, 2 and 14 h after treatment. Hepatocytes were obtained from two animals treated with dimethylnitrosamine at 4 mg/kg (2 h sampling time) and two animals treated with 2-acetylaminofluorene at 50 mg/kg (14 h sampling time). Hepatocytes were isolated, cultured and labelled with [methyl-3H]thymidine specific activity 79-83 Ci/mmol) from which autoradiographs were prepared and analysed.


Results were subjected to statistical analysis using classical one-way analysis of variance followed by a Student's t-test.  A positive response is normally indicated by a substantial and dose-related statistically significant increase in both the gross and net nuclear grain counts compared with the concurrent control values.


 


Results


Clinical signs including lethargy, unsteady gait and piloerection were seen within 30 min of administration of high levels of bromoform confirming absorption of the compound following oral administration. These signs gradually moderated over a period of up to 4 days (depending on the dosage).


 


Bromoform did not caused any significant increases in the gross or net nuclear grain counts at either sampling time.


 


The positive control agents, dimethylnitrosamine and 2-acetylaminofluorene both caused highly statistically significant increases in both gross and net nuclear grain counts in each experiment.


 


Conclusion


Bromoform did not show any DNA damaging activity in the rat liver UDS assay and is considered negative for genotoxicity in this assay.