Bromoacetic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January to July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EPA, etc)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

a nd EU method B.11

Deviations:

yes

Remarks:

only males were used because no sex differences were found in a previous chromosomal aberration study performed with Bromoacetic acid (see TNO report V 90.215). In this study cytotoxicity was found in female rats.

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Bromoacetic acid

EC Number:

201-175-8

EC Name:

Bromoacetic acid

Cas Number:

79-08-3

Molecular formula:

C2H3BrO2

IUPAC Name:

2-bromoacetic acid

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Bromoacetic acid
- Substance type: White to yellow crystals
- Physical state: Solid
- Analytical purity: 96.6%
- Lot/batch No.: S4394760
- Stability under test conditions: Stable at room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 151.5-179.3 g
- Assigned to test groups randomly: yes, under following basis: computer randomization
- Housing: The rats were housed with five per cage under conventional conditions in one room in sterilised macrolon cages (type IV), fitted with a grid cover of stainless steel and with a bedding od wood shavings (Lignocel). Enviro-dry was used as cage enrichment.
- Diet (e.g. ad libitum): ad libitum from the arrival of the animals until the end of the study.
- Water (e.g. ad libitum): ad libitum from the arrival of the animals until the end of the study.
- Acclimation period: yes, during this period the animals were observed daily for overt signs of ill health and anomalies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-3°C
- Humidity (%): 40% and not exceeding 70%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Controls: Yes
- Vehicle: Injection water

Details on exposure:

DOSE RANGE FINDINGS STUDY was performed - step-wise approach
- Dosing volume of the test substance (20 ml/kg-bw).
- Observation: 1 h, 4 and 24 hours post treatment.
PREPARATION OF DOSING SOLUTIONS and DIET PREPARATION
Details not reported

Duration of treatment / exposure:

Sacrifice:
- Group A (negative control) 24 h (5 males) and 48 h (5 males)
- Group B (monobromoacetic acid): 50 dose level (mg/kg bw) - 24 h (5 males)
- Group c (monobromoacetic acid): 100 dose level (mg/kg bw) - 24 h (5 males)
- Group D (monobromoacetic acid): 150 dose level (mg/kg bw) - 24 h (5 males) and 48 h (5 males)
- Group E (mytomycin C - positive control): 3.0 dose level (mg/kg bw) - 24 h (5 males)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 50, 100, 150 mg/kg bw
Basis:
nominal in diet
monobromoacetic acid

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3.0 mg/kg bw
Basis:
nominal conc.
intraperitoneal injection of Mitomycin C

No. of animals per sex per dose:

10 animals for control and high-dose, 5 animals for low- and mid-dose

Control animals:

yes

Positive control(s):

- Substance used as Positive Control: Mitomycin C, 3.0 mg/kg bw
- Vehicle: Positive: control by intraperitoneal injection

Examinations

Tissues and cell types examined:

- Clinical signs: Yes - 4 hours post-treatment
- Tissue: Bone marrow
- Number of animals: 5 animals/sex/group at each time point
- Number of cells: 20, 100, 200, 1000, 2000 or other
- Time points: 6, 24, 48 h after treatment
- Type of cells: bone marrow
- Parameters: numbers and types of structural aberrations and mitotic index

Details of tissue and slide preparation:

After sacrifice, the femurs were dissected free of all adherent muscle and tissue. Bone marrow cells were removed from the femurs by flushing with Hank's balanced salt solution (HBBS), exposed to a hypotonic solution (0,075 M potassium chloride, pre-warmed to 37 °C), fixed in freshly prepared 3: 1 (v/v) mixture of methanol and glacial acetic acid and processed for chromosomal preparations.

The slide, were stained with a 2% Giemsa (Merck-Darmstadt, Darmstadt, Germany) solution, air-dried and embedded. Two slides per animal were prepared for both the mitotic index scoring and chromosomal aberration analysis. Two slides per animal were examined, One thousand cells, 500 cell per slide), were examined to determine the percentage of cells in mitosis (mitotic index). Of each animal, 100 well-spread metaphases (5-0 metaphases per slide), each containing 40-42 centromeres, were analysed by microscopic examination for both chromatid-type and chromosome-type aberration, and other anomalies such as endoreduplicated cells, polyploid cells or heavily damaged cells. Endoreduplicated cells, polyploid cells, and heavily damaged cells were recorded but not included in the 100 analysed cells per animal. The Vernier readings of all aberrant metaphase, observed were recorded.

Evaluation criteria:

The Vernier readings of all aberrant metaphase

Statistics:

Data were analyzed by Analysis of Variance (ANOVA), after square root transformation (sqrt(x+1)) to 'normalise' the distribution of the counts (Lovell et al., 1989). If the ANOVA yielded significant results, pairwise comparisons between treated and control groups were made. Data were analyzed statistically by non-parametric Kruskal-Wallis ANOVA. At the time point 24 hours, data from groups A,B,C and D were subjected to Kruskal-Wallis one way ANOVA. Kruskal-Wallis ANOVA was applied to the negative control group A versus treatment groups B,C and D.
All statistical tests were performed using BMDP statistical software (W.J Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the high-dose group (150 mg/kg bw ), 7 out of 10 rats were found dead within 24 hours after treatment. It was decided to treat the two reserve
rats with a dose level of 125 mg/kg bw . These two rats also died after treatment. Because not enough surviving rats remained for both scheduled sacrifice time points, it was decided to sacrifice the 3 surviving rats of the high-dose group (150 mg/kg bw ) at 24 hours. The 5 negative
(injection water) control rats, assigned to sacrifice time point 48 hours, were euthanized, but not used for further analysis.

1) Dose range finding:
In the dose range finding toxicity test, dose levels between 50 and 300 mg/kg bw were administered to groups of 3 animals in a stepwise manner. Rats of the control group were treated in a similar way with the vehicle (injection water) only. Twenty-four hours after treatment, all animals were sacrificed and the mitotic index in bone marrow cells was determined. Severe clinical signs and mortality were found at 200 mg/kg bw and above. Dose levels of 50, 100 and 150 mg Bromoacetic acid/kg bw were selected for the chromosomal aberration study.

2) Chromosomal aberration study:
Compared to the control, a decrease in the mean mitotic index was observed at all dose levels analysed (50, 100 and 150 mg/kg bw), statistically significant at 50 and 150 mg/kg bw. This demonstrates that the test substance or metabolites thereof have reached the bone marrow and induced cytotoxicity to the bone marrow cells. None of the dose levels of the test substance (50, 100 and 150 mg/kg bw) induced a statistically significant increase in the number of cells with structural chromosomal aberrations, when compared to the concurrent negative control (injection water). This indicates that treatment with Bromoacetic acid does not result in clastogenicity to bone marrow cells of rats. The positive control substance mitomycin C induced the expected statistically significant increase in the incidence of structural chromosomal aberrations, demonstrating the validity of the test system.
From the results obtained in this in-vivo chromosomal aberration test, it is concluded that Bromoacetic acid, close to the lethal dose, was cytotoxic
to the bone marrow, but did not induce structural chromosomal aberrations in the bone marrow cells of male rats, under the conditions used in this study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
From the results obtained in this in-vivo chromosomal aberration test, it is concluded that Bromoacetic acid, close to the lethal dose, reached the bone marrow and induced cytotoxicity to the bone marrow cells.
Bromoacetic acid did not induce structural chromosome aberrations in bone marrow cells of male rats under the conditions employed in this study.