2-bromo-5-hydroxy-4-methoxybenzaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-11-27 to 1998-02-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

only 4 strains were tested (did not include an E. coli strain or S. typhimurium TA102); an expert statement is added to justify no further testing

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

four of the five recommended strains were tested

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

four of the five recommended strains were tested

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate issued by the Federal Republic of Germany

Type of assay:

bacterial reverse mutation assay

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 specified

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

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver homogenate S9

Test concentrations with justification for top dose:

- Pre-Experiment (TA98 and TA100, with and without S9): 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate;
- Experiments 1 and 2 (with and without S9): 10, 33, 100, 333, 1000 and 2500 µg/plate;

Since the test item was soluble in the solvent (DMSO) up to the standard limit concentration recommended in the regulatory guidelines that this assay followed (5000 µg/plate), the highest tested concentration in the Pre-Experiment was 5000 µg/plate.
The highest tested concentration in Experiments 1 and 2 were selected based on the toxicity of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9, 10 µg/plate (TA1535 and TA100)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without S9, 10 µg/plate (TA98), 50 µg/plate (TA1537)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9; 2.5 µg/plate (TA1535, TA1537, TA98 and TA100)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1- in agar (plate incorporation):
In the plate incorporation assay, the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation); 100 µL Bacteria suspension (cf. test system, pre-culture ofthe strains); 2000 µL Overlay agar.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
- Experiment 2- preincubation:
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°Cfor 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

DURATION
- Preincubation period: 60 min (Experiment 2)
- Exposure duration: at least 48 hours (Experiments1 and 2)
- Selection time: at least 48 hours (simultaneous with exposure)
- Fixation time: at least 48 hours

SELECTION AGENT:
-histidine

NUMBER OF REPLICATIONS: triplicate
For each strain and dose level, including the controls three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

OTHER:
-The colonies were counted using the AUTOCOUNT that was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the background growth the colonies were counted manually from 333 ug/plate up to the highest concentration in the presence of S9 mix and at 2500 without S9 mix.

Rationale for test conditions:

Solubility limitations: Since the test item was fully soluble in DMSO, the highest tested concentration for the pre-experiment was 5000 μg/plate.

Evaluation criteria:

- The test substance is considered positive if either a reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
- A test substance producing neither a reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
- The test substance is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
- Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate and upwards (without S9); at 333 µg/plate and upwards (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate (without S9); at 333 µg/plate and upwards (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity ofthe test article a pre-experiment was performed with strains TA98 and TA 100. Eight concentrations (3-5000 µg/plate) were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). As the results of the pre-experiment were in accordance with the acceptability criteria, the pre-experiment is reported as a part of the main experiment.
Based on the results of the pre-experiment, 2500 µg/plate was selected as the highest test item concentration for Experiment 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
- no data; Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Remarks on result:

other: Experiment 1

Any other information on results incl. tables

Cytotoxic effects in the main experiment:

Strains	Experiment 1 (ug/plate)	Experiment 1 (ug/plate)	Experiment 2 (ug/plate)	Experiment 2 (ug/plate)
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 1535	2500	/	/	1000 -2500
TA 1537	2500	333 -2500	2500	333 -2500
TA 98	2500	333 -2500	1000 -2500	1000 -2500
TA 100	2500	333 -2500	1000 -2500	333 -2500

Toxic effects, evident as a reduction in the number of revertants, occurred in the test groups at the concentrations listed in the table above. The background growth was reduced at the higher concentrations with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the four tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The positive controls showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.