2-bromo-5-hydroxy-4-methoxybenzaldehyde

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-04-22 to 1998-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

However deviations from the protocol are reported in seciton "overall remarks

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

However deviations from the protocol are reported in seciton "overall remarks

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

426-540-0

EC Name:

-

Cas Number:

2973-59-3

Molecular formula:

C8 H7 Br O3

IUPAC Name:

2-bromo-5-hydroxy-4-methoxybenzaldehyde

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00265786
- Expiration date of the lot/batch: July 01, 1998 (Retest date)
- Purity test date: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated by the sponsor

FORM AS APPLIED IN THE TEST (if different from that of starting material): formulated in polyethylene glycol (PEG 400)

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 FUllinsdorf
- Age at study initiation: 8-12 weeks (at the start of acclimation)
- Weight at study initiation: males mean value 35.0 g (SD ±3.3 g); females mean value 26.8 g (SD ±2.0 g)
- Assigned to test groups randomly: yes
- Fasting period before study: Approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: Animals were housed individually in Makrolon Type I cages, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Humidity (%): 23-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (PEG 400)
- Justification for choice of solvent/vehicle: The vehicle was chosen based on its relative non-toxicity for the animals.
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): 9726 (gas chromatography grade)
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- On the day of the experiment, the test substance was formulated in PEG 400. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume (10 mL/kg bw) to be administered was adjusted to the animal’s body weight.

DIET PREPARATION
- Rate of preparation of diet (frequency): not applicable
- Mixing appropriate amounts with (Type of food): not applicable
- Storage temperature of food: not applicable

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 h and 48 h preparation interval

No. of animals per sex per dose:

six males and six females were treated per dose group and sampling time.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (dissolved in deionisd water)
- Justification for choice of positive control(s): The study does not provide a justification for the choice of positive control. However, cyclophosphamide is one of the recommended positive control choices in OECD Guideline 474, which this study follows.
- Route of administration: oral: gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow -at least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test articles.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Approximately 18 hours before treatment the animals received no food but water ad libitum.
At the beginning ofthe treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling ofthe bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop ofthe resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation ofthe slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic eiythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described.

Evaluation criteria:

- A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
- A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

- Statistical significance was measured by means of the nonparametric Mann-Whitney test. Both biological and statistical significance were considered together.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The animals expressed slight toxic reactions in the assay (1 out o 12 females treated with 20000 mg/kg bw died)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg only
- Solubility: no data
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity was observed in both males and females at 1, 6, 24 and 48 hours. Eyelid closure was at 1 hour in one female and at 6 hours in 1 male and 1 female. Apathy was observed in 1 female at 6 hours.
- Evidence of cytotoxicity in tissue analyzed: not examined
- Rationale for exposure: no data
- Harvest times: not applicable
- High dose with and without activation: On the basis of these data 2000 mg/kg bw were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed were near to the range of the vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean value of NCEs of the corresponding vehicle controls indicating that the test substance had no cytotoxic properties in the bone marrow.
- Appropriateness of dose levels and route: As estimated by a pre-experiment a dose of 2000 mg/kg bw was suitable. The animals expressed slight toxic reactions. In the micronucleus assay 1 out of 12 females treated with this dose died.
- Statistical evaluation: 40 mg/kg b.w. cyclophosphamide (positive control) administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency (p<0.0001).

Any other information on results incl. tables

Pre-Experiment results:

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. T002019 formulated in PEG 400. The volume administered was 10 mI/kg b.w.

The treated animals expressed toxic reactions as shown in the table (left number : malse; right number : females):

Toxic Reactions	1 h	6 h	24 h	48 h
reduction ofspontaneous activity	1/1	2/2	2/2	1/1
eyelid closure	0/1	1/1	0/0	0/0
apathy	0/0	0/1	0/0	0/0

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

Definitive Study:

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE / NCE
Vehicle	0	24	0.080	0 - 3	20000 / 2139
Test Article	200	24	0.110	0 - 5	2000 / 1620
Test Article	670	24	0.085	0 - 4	2000 / 1560
Test Article	2000	24	0.085	0 - 5	2000 / 1686
Cyclophosphamide	40	24	1.900	25 - 63	2000 / 1833
Test Article	2000	48	0.070	0 - 5	2000 / 1645

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of orally treated mice. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.