N'-(1,3-dimethylbutylidene)-3-hydroxy-2-naphthohydrazide

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:

03 July 1998 - 14 July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: see "Principles of method if other than guideline"

Version / remarks:

Similair to OECD TG 471

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

- According to the method described in "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances' (Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW & No. 2 (1997) ofthe Basic Industries Bureau, MITI, October 31, 1997).

- Deviations: No justification for the use of duplicate test cultures instead of triplicate test cultures

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Substance type: Light brown crystal
- Physical state: solid
- Storage condition of test material: stored in a cold dark place

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced with phenobarbital and 5,6-benzoflavone
- Composition of S9 mix: S9-mix per mL: 4 μmol NADPH, 4 μmol NADH 5 μmol glucose-6-phosphate; 100 μmol sodium phosphate buffer pH 7.4; 8 μmol MgCl2; 33 μmol KCl and 0.1 mL of S9

Test concentrations with justification for top dose:

Dose range finding test:
S. typhimurium TA100, TA98, TA1535, TA1537 and E. coli WP2 uvr A: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate in the absence and presence of S9-mix.

Main Experiment (without S9-mix):
S. typhimurium TA100 and TA1535: 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate
S. typhimurium TA98, TA1537 and E. coli WP2 uvr A: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

Main Experiment (with S9-mix):
S. typhimurium TA100,TA1535 TA98, TA1537 and E. coli WP2 uvr A: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

Justification for Top dose: Limit of cytotoxicity

Vehicle / solvent:

- Vehicle used: DMSO
- The Substance was stable and dissolved in DMSO (no change in the 50 mg/mL solution prepared with dehydrated DMSO, including such as color change and heat generation, until 2 hours after preparation was observed)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide)

Remarks:

In DMSO; Without S9: TA100 (0.01 µg/plate); WP2 uvr A (0.01 µg/plate); TA98 (0.1 µg/plate)

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In distilled water; Without S9: TA1535 (0.5 µg/plate)

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191 (2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl)

Remarks:

In DMSO; Without S9: TA1537 (1 µg/plate)

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AA (2-Aminoanthracene)

Remarks:

In DMSO: With S9: TA100 (1 µg/plate), TA1535 (2 µg/plate), TA98 (0.5 µg/plate), TA1537 (2 µg/plate) and E. coli WP2 uvr A (10 µg/plate)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Positive control and test substance concentrations: duplicate
Negative control: Triplicate

- Number of independent experiments: 2 (dose range finder and main experiment)

METHOD OF TREATMENT/ EXPOSURE:
In agar (plate incorporation); preincubation


TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

COLONY COUNTING
The number of revertant colonies was counted using the manual counter or the colony analyzer (CA-7, Toyo-sokki Co., Ltd). With the colony analyzer, the count was finalized after correcting for area and count drop. Each plate was measured twice, and the average of these two measurements was adopted as the number of revertant colonies on the plate.

NUMBER OF CELLS EVALUATED: > 2E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: background growth inhibition
- Other: precipitation of the test item was recorded by visual inspection.

Evaluation criteria:

The test substance is considered positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner given the results are reproducible. When these conditions are not met the substance is considered negative.

Statistics:

Not applicable.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

RANGE-FINDING/SCREENING STUDIES:
Precipitation of the test item on the plates was observed at concentrations of 1250 µg/plate and higher. In the dose-range finding study growth was inhibited at 313 µg/plate and above in TA100 and TA1535 and at 1,250 µg/plate and above in WP2 uvrA, TA98 and TA 1537 in the absence of S9 mix. Growth was inhibited in all tester strains at 1250 µg/plate and higher in the presence of S9 mix.
Based on the results of the dose range finding test, 625 µg/plate of strains of TA100 and TA1535, and 1,250 µg/plate for strains of WP2 uvrA, TA98 and TA1537 in the absence of S9 mix, and 1,250 µg/plate for all of the tester strains in the presence of S9 mix were determined as the highest dose for the main test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls induced significant increase in the number of revertant colonies and the number of revertant colonies in the positive controls and the negative control was within the range of the historical laboratory background data. These results confirm that the test conditions were adequate and that the metabolic activation system functioned properly.
Ames test:
- Signs of toxicity : In the main study growth was inhibited at 313 µg/plate for TA100 and TA1535, at 625 µg/plate for TA98 and TA1537 and 1250 µg/plate for WP2 uvrA in the absence of S9. Growth inhibition was observed at 1,250 ug/plate in all of the test strains in the presence of S9 mix.
- Mean number of revertant colonies per plate and standard deviation : The number of revertant colonies for all the test strains was less than twice that of the negative control with and without S9 mix. (see attached background material for detailed results)

Applicant's summary and conclusion

Conclusions:

Based on the results of an Ames test performed according to a guideline similair to OECD 471 and in accordance with GLP principles, BMH is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was performed according to the method described in : Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances"; similair to OECD 471 and in accordance with GLP principles. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate. Cell growth was inhibited at 313 µg/plate and above in TA100 and TA1535 and at 1,250 µg/plate and above in WP2 uvrA and at 625 µg/plate and above in TA98 and TA 1537 in the absence of S9 mix. Growth was inhibited in all tester strains at 1250 µg/plate and higher in the presence of S9 mix. Precipitation of the test item on the plates was observed at concentrations of 1250 µg/plate and higher. Based on the results of the dose range finding test, 625 µg/plate for strains of TA100 and TA1535, and 1,250 µg/plate for strains of WP2 uvrA, TA98 and TA1537 in the absence of S9 mix, and 1,250 µg/plate for all of the tester strains in the presence of S9 mix were determined as the highest doses for the main test. In the main study growth was inhibited at 313 µg/plate for TA100 and TA1535,  at 625 µg/plate for TA98 and TA1537 and 1250 µg/plate for WP2 uvrA in the absence of S9. Growth inhibition was observed at 1,250 ug/plate in all of the test strains in the presence of S9 mix. Positive and negative controls were included and showed that the test system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant colonies in the tester strains (TA1535, TA1537, TA98 and TA100 and WP₂uvrA) both in the absence and presence of S9 -metabolic activation when tested up to the limit of cytotoxicity. Based on the results of this study it is concluded that BMH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay.