Isophthalohydrazide

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:

09 November 2017 - 06 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isophthalohydrazide

EC Number:

220-425-7

EC Name:

Isophthalohydrazide

Cas Number:

2760-98-7

Molecular formula:

C8H10N4O2

IUPAC Name:

isophthalohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystal powder
Test item storage: At room temperature

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:

5% rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

Experiment 1:
TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plateµg/plate
Experiment 2:
TA1535, TA1537 and TA98, TA100 and WP2uvrA
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plateµg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle:
Test item dissolved in dimethylsulfoxide and has been accepted and approved by authorities and international guidelines.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

without S9 (Concentrations/solvent:See table 1)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9 (Concentrations/solvent:See table 2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: first experiment: direct plate; second experiment: pre-incubation

DURATION
- Exposure duration: 48 hour ± 4 h

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments, a direct plate assay and a pre-incubation assay, were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were determined both macroscopically and microscopically by using a dissecting microscope.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or withoutS9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

INTERPRETATION
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of IDH on the plates was not observed at the start or at the end of the incubation period in both experiments.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed in all tester strains in the absence and presence of S9-mix in both experiments


COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON MUTAGENICITY:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested. Except for tester strain WP2uvrA in the absence of S9-mix at concentration of 5000 μg/plate. The test item induced a 2.1 -fold increase in the number of revertant colonies compared to the solvent control at the highest tested dose level of 5000 μg/plate. The increase observed was well within the historical control data range and therefore considered not biologically relevant.

Applicant's summary and conclusion

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, IDH was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that IDH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.