Adipohydrazide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese: Test data for registration of agricultural chemicals. Guideline 2-1-19-1

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

adipic dihydrazide

IUPAC Name:

adipic dihydrazide

Constituent 2

Reference substance name:

ADH

IUPAC Name:

ADH

Details on test material:

Appearance: White crystalline powderStorage conditions: Room temperature in the darkLot number: 0E96Purity: > 99%Expiry date: 25 May 2012Date received: 04 October 2010

Method

Target gene:

Salmonella typhimurium TA1535 hisG46 rfa deletion uvrBSalmonella typhimurium TA1537 hisC3076 rfa deletion uvrBSalmonella typhimurium TA98 hisD3052 rfa deletion uvrB pKM101Salmonella typhimurium TA100 hisG46 rfa deletion uvrB pKM101Escherichia coli WP2 uvrA (pKM101) trpE ochre uvrA pKM101

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

nitroreductase deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C.

Test concentrations with justification for top dose:

5 to 7 concentrations up to 5000 µg/plate.

Vehicle / solvent:

Water.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive control substances in the absence of S9 mix:Sodium azide for strains TA100 and TA15359-Aminoacridine for strain TA15372-Nitrofluorene for strain TA984-Nitroquinoline-1-oxide for strain WP2 uvrA (pKM101)Positive control substances in the presence of S9 mix:2-Aminoanthracene for strains TA100 and TA1535; WP2 uvrA (pKM101)Benzo[a]pyrene for strains TA98 and TA1537Procedure - First test:Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass vessels. The vehicle control was water. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained.Procedure - Second test:As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system.If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. Biological importance should always beconsidered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.The statistical procedures used in some cases are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

First test:No evidence of toxicity was obtained following exposure to ADH. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ADH at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.Second test:No evidence of toxicity was obtained following exposure to ADH.No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ADH at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For individual and mean results, see the attachment.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negativeAdipic acid dihydrazide showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

An in vitro assessment of the mutagenic potential of adipic acid dihydrazide in histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), was performed according to EU- and OECD-methods.

 

No evidence of toxicity was obtained following exposure to adipic acid dihydrazide.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to adipic acid dihydrazide at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Adipic acid dihydrazide showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.