Bromoform

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity of the test substance in Ames, Mouse Lymhoma and Sister Chromatid Exchange Assays in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company.
- Purity: 97.7%

Target gene:

- S. typhimurium: His-locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced Sprague-Dawley rats or Syrian hamsters liver S9 (due to the nature of the study as an inter-laboratory trial, tests were run using both types).

Test concentrations with justification for top dose:

For the standard plate method: 0, 100, 3.333, 1000, 6,666, 10,000 µg/mL

For the desiccator method designed to take account of the volatility of the test substance: 0.000, 0.001, 0.005, 0.007, 0.010, 0.020, 0.025, 0.035, 0.050, 0.100, 0.500, 1.000 mL/chamber.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine

Evaluation criteria:

Evaluations of the results were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or non-mutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose-response. A trial was questionable (?) if the dose response was judged insufficiently high to support a call of "+ W", if only a single dose was elevated over the control, or if a weak increase was not dose-related.

The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach twofold over background for a trial to be judged "+" or "+ W". A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it gave a reproducible dose-related response over the solvent control in replicate trials, and judged questionable (?) if the results of individual trials were not reproducible or if only single doses produced increases in his' revertants in repeat trials. Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Remarks:

No vehicle used

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Remarks:

No vehicle used

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Remarks:

No vehicle used

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: TA97, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: desiccator method

Conclusions:

The test substance was considered positive for mutagenicity in salmonella typhimurium in the Ames test.

Executive summary:

Introduction

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 Mutation Test

Method

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 S9.

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.

 

Results

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.

Conclusion

The test substance was considered positive for mutagenicity in salmonella typhimurium the Ames test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Version / remarks:

The study was part of an inter-laboratory trial using two laboratories. Only the results from one laboratory are reported in this study summary as the test in the second laboratory was negative. However, this may have been due to the lower top dose used and hence the relability of the result from the second laboratory is uncertain.

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Aldrich Chemical Company.
- Purity: 97.7%

Further specific information on the test material is in Zeiger E. (1990): Mutagenicity of 42 chemicals in Salmonella. Environ Molec Mutagen 16(Suppl 18):32-54.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells were cloned at Litton Bionetics Inc. and supplied to the other laboratories. Cells were not used beyond 15 passages after cloning. Cells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% foetal calf serum at 37°C using 5% CO2. Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from male Sprague-Dawley rats induced with Aroclor 1254.  

Test concentrations with justification for top dose:

The study was part of an inter-laboratory trial. Only the results from one laboratory are reported in this study summary (see additional information on results section).

Laboratory One (laboratory to which the results apply)
0, 29, 96, 290 µg/mL with S9
0, 96, 290 and 968 µg/mL without S9

Doses were selected based on trial experiments designed optimse dose levels.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Evaluation criteria:

Data were evaluated for both trend and dose point increase over the solvent control. A trend of P < 0.005 or an individual dose with a 20% increase over the solvent control was considered significant.

If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+ W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the chromsome aberrations trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+ W*). The term “weak positive” (+ W) refers to the strength of the evidence for the positive call, not to the potency of the response.

In general, positive responses were repeated, whereas repeats were not required for the trials concluded to be negative. If positive responses were obtained both with and without S9, the laboratories were required to repeat only one activation condition. When combining the conclusions of individual trials, more emphasis was given to trials in which experimental conditions were optimised. For example, a trial that included an extended harvest time was generally given more weight, whereas a trial that had a positive response only at doses judged to be toxic was given less weight. However, when trials were considered to be equivalent the following scheme was used in combining trial calls (+) and (-) = (?); (+) and (+W) or (?) = (+); (+W) and (?) = (+ W); (-) and (+ W) or (?) = (-); (W*) was considered to be equivalent to a (+) when combining calls.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

other: weak positive

Cytotoxicity / choice of top concentrations:

other: For toxic compounds, cells from the highest dose likely to yield analysable metaphases were fixed, together with cells from five successively lower dose levels.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: For toxic compounds, cells from the highest dose likely to yield analysable metaphases were fixed, together with cells from five successively lower dose levels.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Testing was undertaken as an inter-laboratory trial with testing being undertaken at two laboratories. The maximum dose tested was higher at one laboratory which observed a weak positive response in the test. The other laboratory tested at lower doses and obtained negative results in both the presence and absence of metabolic activation. The results from this laboratory have not been included as they are not considered relevant given the results from the laboratory which tested at a higher concentration.

Remarks on result:

other: The view of the literature paper authors was that this weak positive result needed confirmation.

Conclusions:

The test substance produced a weak positive result in the sister chromatid exchange assay in the avsence of metabolic activation.

Executive summary:

Introduction

The 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.

Method

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 toxicity.

 

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.

Results and Conclusion

The test substance produced a weak positive result in the sister chromatid exchange assay in the absence of metabolic activation.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

not applicable

GLP compliance:

no

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Test chemicals used for the study were supplied coded by the National Toxicology Program's (NTP) chemical repository (Radian Corporation, Austin, Tex). The original source and purity are in Zeiger E, Haseman J. Shelby M. Margolin B, Tennant R (1990): Evaluation of Four In Vitro Genetic Toxicity Tests for Predicting Rodent Carcinogenicity: Confirmation of Earlier Results with 41 Additional Chemicals. Environ Mol Mutagen 16(Suppl 18): 1-14.

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Hepatic S9 mix prepared from Aroclor 1254-induced male Fischer 344 rats.

Evaluation criteria:

Solvent control cultures
1. The cloning efficiency must be in the 50-120% range.
2. At least two acceptable cultures must be available.
3. The average mutant frequency for all acceptable cultures must be between 15 x 10^-6 and 110 x 10^-6 for negative evaluations. For mutagenic test chemicals the range is extended to 10 x 10^-6 to 150 x 10^-6.
4. A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at P ≤ 0.05.

Positive control cultures
1. The cloning efficiency must be in the 10-120% range.
2. The relative total growth must not be less than 1%.
3. At least one acceptable culture must be available.
4. The average mutant frequency for all acceptable cultures must be within the historical range


Test chemical cultures
1. The cloning efficiency must be in the 10-120% range.
2. The relative total growth must not be less than 1%.
3. The relative suspension growth for the second day of expression must be 40% or greater
4. The test chemical must remain soluble during treatment.
5. The maximum dose is 5 mg/ml for solids and 5 µg/ml for liquids.
6. Each dose level must have two or more acceptable cultures.
7. A Chi-square test for consistency among the mutant frequencies of the acceptable cultures must be significant at P ≤ 0.05.
8. At least three acceptable dose sets must be available, except when no response is obtained and sets are rejected due to precipitation.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: Publication states that the high dose was determined by solubility or cytotoxcity, but did not exceed 5 mg/mL.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Publication states that the high dose was determined by solubility or cytotoxcity, but did not exceed 5 mg/mL.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

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.

Executive summary:

Introduction

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).

 

Method

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 (TFT^r) 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.

 

Results and Conclusion

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

Bromoform (CAS 75-25-2, >99% pure) was obtained from Aldrich Chemical Co., Gillingham, UK.

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

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.

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

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).

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

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.

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.

 

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

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:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Bromoform (CAS 75-25-2, >99% pure) was obtained from Aldrich Chemical Co., Gillingham, UK.

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CD1 outbred albino mice of Swiss origin were obtained from Charles River, UK
- Age at study initiation: approx. 35 days old on disptach from the supplier
- Weight at study initiation: 22-24g on dispatch from the supplier.
- Assigned to test groups randomly: Not stated.
- Fasting period before study: Yes
- Housing: Animals were group-housed in polystyrene disposable cages containing certified contaminant free woodchip bedding.
- Diet (e.g. ad libitum): ad libitum, except overnight prior to, and for 2 h after, treatment.
- Water (e.g. ad libitum): ad libitum, except overnight prior to, and for 2 h after, treatment.
- 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

Route of administration:

oral: gavage

Vehicle:

aqueous 1% w/v methylcellulose

Duration of treatment / exposure:

Single dose of Control, 250, 500 and 1,000 mg/kg at time 0, with sampling at 24 and 48 hours.

Frequency of treatment:

Once

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Vehicle Control

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 male/ 5 female per control and for each dose level at each time point (24 and 48 hours)

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C at 12 mg/kg body weight

Statistics:

Results were analysed using appropriate non-parametric statistical analyses based on rank.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs, including lethargy and staggering, were evident in the high dose group shortly after treatment.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 probably well 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 it is our experience that this is a poor indicator of target organ exposure except for cytostatic or cytotoxic compounds which, in any case, are usually positive in the micronucleus test.

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.

Bromoform did not cause any significant increases in the incidence of micronucleated polychromatic erythrocytes at any dose level or sampling time. Mice treated with 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.

Conclusions:

Bromoform did not show any DNA damaging activity in the mouse micronucleus test and is considered negative for genotoxicity in this assay.

Executive summary:

Introduction

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.

 

Method

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.

 

Results

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.

 

Conclusion

Bromoform did not show any DNA damaging activity in the mouse micronucleus test and is considered negative for genotoxicity in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)