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EC number: 200-854-6
CAS number: 75-25-2
Mutagenicity of the test substance in Ames, Mouse
Lymhoma and Sister Chromatid Exchange Assays in vitro.
The study was conducted to determine the
mutagenic potential of the test substance to Salmonella typhimurium
using a test method comparable to the OECD 471 Bacterial Reverse
S. typhimurium strains TA97, TA98, TA100,
TA1535, and TA1537 were used. Because the testing was done as part of
an inter-laboratory trial testing was conducted in more than one
laboratory on coded samples. The test used a preincubation procedure
with and without exogenous metabolic activation. The metabolising
system was liver S-9 derived from Aroclor 1254-induced Sprague-Dawley
rats and Syrian hamsters. Experiments were conducted with both types of
The test substance is a volatile chemical
and following testing in the preincubation procedure it produced either
negative or equivocal responses in some laboratories. It was therefore
retested at one laboratory using exposure in a desiccator. Test strains
were TA98, TA100, TA1535 and TA1537. In this procedure, S-9 or buffer
was incorporated into the top agar and poured onto the plate. The lids
of the plates were removed and the plates were stacked on a perforated
porcelain plate in a 9 litre glass desiccator jug containing a magnetic
stirring bar. A measured volume of test chemical in liquid form was
introduced into a watch glass suspended below the porcelain plate, and
the desiccator was sealed and placed on a magnetic stirrer in a 37°C
incubator. After 24 hr, the plates were removed from the desiccator and
incubated at 37°C in air for an additional 24 hr.
In the desiccator protocol which is
considered the most reliable method the test substance was positive for
mutagenicity in strain TA 98 in the absence of S9 and positive in strain
TA100 in the presence of S9.
The test substance was considered positive
for mutagenicity in salmonella typhimurium the Ames test.
study was conducted to determine the potential for the test substance to
induce chromosome aberrations in Chinese Hamster Ovary Cells (CHO) using
a test method comparable to OECD method No. 479: Genetic Toxicology: In
vitro Sister Chromatid Exchange Assay in Mammalian Cells.
Chinese hamster lung cells were used for the
study. Cultures were initiated 24 hours prior to treatment. Treatment was
with or without S9 metabolising system. Concurrent solvent and positive
controls were included. Mitomycin C (MMC) was the positive control in
experiments without S9 and cyclophosphamide (CPA) was used in
experiments with S9.
For the tests without S9 the positive
controls and test chemicals were added and the flasks were incubated at
37°C for 2 hr prior to the addition of bromodeoxyuridine (BrdUrd) (M),
after which the incubation was continued for an additional 24 hr. The
cells were then washed and medium containing BrdUrd and Colcemid was
added to the flasks for an additional 2-2.5 hours incubation period. Twenty-four
hours later Colcemid was added and the cells were incubated for an
additional 2-2.5 hours. At the end of the treatment regimen a visual
estimate of the confluency of each flask was made in order to evaluate
At the end of treatment cells were harvested
and resuspended in fixative fixed before slides were prepared and
stained with Hoechst 33258 and scored for sister chromatid exchanges.
The test substance produced a weak positive
result in the sister chromatid exchange assay in the absence of metabolic
The mutagenic potential of the test
substance to L5178Y Mouse Lymphoma Cells was Investigated using
a test method similar to the OECD 490 (In Vitro Mammalian Cell Gene
Mutation Tests Using the Thymidine Kinase Gene).
The highest dose of the compound tested was
determined by solubility or toxicity but did not exceed 5 mg/ml. All
doses were tested at least in duplicate. Cells (6 x 106) were
treated for 4 hr at 37°C, washed, suspended in medium, and incubated for
48 hr at 37°C. After expression, 3 x 106 cells were
plated in medium and soft agar supplemented with trifluorothymidine
(TFT) for selection of TFT resistant (TFTr) cells, and 600
cells were plated in nonselective medium and soft agar to determine the
cloning efficiency. After plating, the cells were incubated for 9-12
days at 37°C before colonies were counted on an Artek 880 colony counter
fitted with a 10-turn size discriminator.
The test substance was considered positive
for mutagenicity in mouse lymphoma L5178Y cells in the absence of S9 and
negative in the presence of S9.
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.
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.
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.
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).
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.
Bromoform did not show any DNA damaging activity in the rat liver UDS assay and is considered negative for genotoxicity in this assay.
The in vivo genotoxicity of Bromoform was investigated in the mouse bone marrow micronucleus test using a protocol performed according to the recommendations of the United Kingdom Environmental Mutagen Society (UKEMS) (Richold et ai, 1990; Kennelly et al, 1993) and the most recent draft guidelines of the Organisation for Economic Co-operation and Development (OECD) available at the time of testing.
45 male and 45 female mice were use in the study. The maximum tolerated dose (MTD) for bromoform was determined over a 4-day observation period. The estimated MTD for bromoform in the mouse was 1000 mg/kg. This dose levels were therefore selected as appropriate maxima for use in the main test.
Groups of mice were treated orally by intragastric gavage with a single dose of the vehicle control, bromoform at 250, 500 or 1000 mg/kg body weight or mitomycin C at 12 mg/kg body weight using a standard dose volume of 20 ml/kg body weight.
Five males and five females from the vehicle control and from test substance treated groups were killed by cervical dislocation 24 and 48 h after dosing. The positive control group was killed 24 h after dosing. Both femurs were removed from each animal, and the bone marrow suspensions were prepared in filtered foetal calf serum. Cell suspensions were centrifuged at 300 g for 5 min. The supernatant was removed and the cells were resuspended in 30 µl of fresh foetal calf serum. A 4 µl drop of cell suspension was used to make a bone marrow smear in the conventional manner. The smears were fixed in methanol, air-dried and stained for 10 min in aqueous 10% Gurr's R66 Giemsa. Slides were rinsed in distilled water, differentiated in buffered distilled water (pH 6.8), air-dried and mounted. The slides were examined (under code) by light microscopy using oil immersion optics.
The incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal was determined. The ratio of polychromatic to normochromatic erythrocytes (p/n ratio) for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated normochromatic erythrocytes was also kept. Micronuclei were identified by the following criteria: (i) large enough to discern morphological characteristics; (ii) possess a generally rounded shape with a clearly defined outline; (iii) deeply stained and similar in colour to the nuclei of other cells— not black; (iv) lie in the same focal plane as the cell; (v) lack internal structure, i.e., they are pyknotic.
Results were analysed using appropriate non-parametric statistical analyses based on rank.
Clinical signs, including lethargy and staggering, were evident in the high dose group shortly after treatment. These signs gradually moderated over the next 22 h and indicated that the compound was absorbed and systemically distributed following oral administration.
Although there was no significant decrease in the proportion of immature erythrocytes following treatment of animals with bromoform. A very slightly higher incidence of micronucleated polychromatic erythrocytes (mnp) was obtained for male mice sampled 48 h after treatment with the high dose level of bromoform. Since this was not significantly greater than the values obtained for the males in the concurrent control group (P > 0.05), and the individual and group mean value for the males fell well within the laboratory historical control range it is not considered to be indicative of DNA-damaging activity. The values for males and females were therefore combined in order to maximise the power of statistical analysis and facilitate interpretation.
Overall, Bromoform did not cause any significant increases in the incidence of micronucleated polychromatic erythrocytes at any dose level or sampling time.
Mice treated with the positive control, mitomycin C, showed the expected large increase in the incidence of micronucleated polychromatic erythrocytes together with a slight decrease in the ratio of polychromatic to normochromatic erythrocytes.
Bromoform did not show any DNA damaging activity in the mouse micronucleus test and is considered negative for genotoxicity in this assay.
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