Adipohydrazide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests, one mouse lymphoma test and an in vitro chromosome aberration assay with human lymphocytes are available. In each test a negative (no genetic toxicity) was obtained.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

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

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 / 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.

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.

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'.

For individual and mean results, see the attachment.

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.

 

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2011) were between 14.1 and 15.8.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

First cytogenetic assay: Without and with S9-mix: 10, 100, 333, 1000, 1500 and 1742 μg/ml culture medium (3 h exposure time, 24 h fixation time); selected for scoring of chromosome aberrations: 10, 1000 and 1742 μg/ml.Second cytogenetic assay:Continuously exposure to Adipic acid dihydrazide in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Adipic acid dihydrazide.Dose levels selected:Without S9-mix : 100, 1000, 1500 and 1742 μg/ml culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time).With S9-mix : 100, 1000, 1500 and 1742 μg/ml culture medium (3 h exposure time, 48 h fixation time).Selected for scoring of chromosome aberrations:Without S9-mix : 1000, 1500 and 1742 μg/ml culture medium.With S9-mix : 1000, 1500 and 1742 μg/ml culture medium.

Vehicle / solvent:

Adipic acid dihydrazide was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml. Adipic acid dihydrazide concentrations were used within 2 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Adipic acid dihydrazide was tested in a dose range-finding assay in the absence and in the presence of 1.8% (v/v) S9-fraction. The highest tested concentration was 1742 μg/ml (= 0.01 M).Final test:Adipic acid dihydrazide was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.First cytogenetic assay:Lymphocytes were cultured for 48 h and thereafter exposed in duplicate to selected doses of Adipic acid dihydrazide for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.Second cytogenetic assay:Lymphocytes were cultured for 48 h and thereafter exposed in duplicate to selected doses of Adipic acid dihydrazide for 24 h and 48 h in the absence of S9-mix and for 3 h in the presence of S9-mix.After 3 h exposure, the cells exposed to Adipic acid dihydrazide in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44 - 46 h (48 h fixation time).The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).Appropriate negative and positive controls were included in the second cytogenetic assay.During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/ml medium)

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-square test, one-sided, p < 0.05.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results for both assays:

Both in the absence and presence of S9-mix, Adipic acid dihydrazide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Both in the absence and presence of S9-mix, Adipic acid dihydrazide did not show a biologically relevant increase in the number of polyploid cells and cells with endoreduplicated chromosomes.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.

The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.

It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:

Interpretation of results (migrated information):negativeAdipic acid dihydrazide is not clastogenic in human lymphocytes under the experimental conditions described.

Executive summary:

The possible clastogenicity of Adipic acid dihydrazide was tested in two independent in vitro experiments, according to the OECD and EC guidelines.

In the first cytogenetic assay, Adipic acid dihydrazide was tested up to 1742 μg/ml (= 0.01 M) for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration recommended for testing in the guidelines.

In the second cytogenetic assay, Adipic acid dihydrazide was tested up to 1742 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix Adipic acid dihydrazide was also tested up to 1742 μg/ml for a 3 h exposure time with a 48 h fixation time.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations.

Adipic acid dihydrazide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No biologically relevant effects of Adipic acid dihydrazide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Adipic acid dihydrazide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Adipic acid dihydrazide is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

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:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine- Salmonella typhimurium

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced microsomes of rat liver

Test concentrations with justification for top dose:

62, 185, 556, 1667 and 5000 µg per plate without external metabolisation, and62, 185, 556, 1667 and 5000 µg per plate with an external metabolising system

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water was used as solvent for the test substance and for the negative control group.

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

Positive control substances:- 2-Aminoanthracene- 7,12-Dimethylbenz[a]anthracene- 1,8-Dihydroxy-anthraquinone- 2-Nitrofluorene- Sodium azide- 4-Nitro-o-phenylenediamine- t-Butyl-hydroperoxide1,8-Dihydroxy-anthraquinone, 7,12-dimethylbenz[a]anthracene and 2-aminoanthracene are mutagenic only when activated by a metabolising system; 4-nitro-o-phenylenediamine, 2 nitrofluorene, t butyl-hydroperoxide and sodium azide are directly acting mutagens.

Evaluation criteria:

The criteria for a positive result are: A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants. • For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants. These threshold values were derived from the variations in the control samples of historic data of the Ames test.

Statistics:

Means and standard deviations were calculated for the number of mutants in every concentration group.

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All positive control substances increased the mutation frequency to more than the threshold values stated above. As 2-aminoanthracene, 1,8-dihydroxy anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No precipitation of the test substance was seen in any of the concentration groups.

In the preliminary test and in the main test no toxicity was seen up to 5000 µg/plate.

 

There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

 

The mean results are presented in the attachment.

Conclusions:

Interpretation of results (migrated information):negativeAdipic acid dihydrazide is not genotoxic in the reverse bacterial mutation assay, in each of the 5 strain tested and with and without an external metabolizing system.

Executive summary:

Adipic acid dihydrazide was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14. Adipic acid dihydrazide is not genotoxic in the reverse bacterial mutation assay, in each of the 5 strain tested, and with and without an external metabolizing system.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Experiment Iwithout S9 mix: 112.5 - 225 - 450 - 900 - 1800 μg/mLwith S9 mix: 112.5 - 225 - 450 - 900 - 1800 μg/mLExperiment IIwithout S9 mix: 112.5 - 225 - 450 - 900 - 1800 μg/mLThe assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

Vehicle / solvent:

Deionised water.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Without metabolic activation: methyl methane sulfonate. With metabolic activation: cyclophosphamideMigrated to IUCLID6: and methyl methane sulfonate

Details on test system and experimental conditions:

A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.1x10E7 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x10E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector.The highest concentration used in the pre-test was chosen with regard to the purity (99.5 %) and the molecular weight of the test item (174 g/mol). Test item concentrations between 14.1 and 1800 μg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Following 4 h (with and without metabolic activation) and 24 h treatment (without metabolic activation) no relevant toxic effects were observed up to the maximum concentration. Therefore, the highest concentration applied to the main experiments was 1800 μg/mL corresponding to about 10 mM as well. The lower concentrations were separated by a factor of 2.0.In the pre-experiment there was no relevant shift of osmolarity and pH-value of the medium even in the stock solution of the test item.In the mutation experiment 1×10E7 cells/flask (80 cm² flasks) suspended in 10 ml RPMI medium with 3 % horse serum (15 % horse serum in the second experiment) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of 48 h.The cell density was determined each day and adjusted to 3×10E5 cells/ml, if necessary.The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector (1). One sample of the cells was taken at the end of treatment (4 and 24 h, respectively), diluted and seeded into microtiter plates, to determine the viability of the cells after treatment (cloning efficiency 1).After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×10E3 cells in selective medium (see below) with TFT (SERVA, D-69042 Heidelberg). The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

Evaluation criteria:

The numbers of colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).The survival rate and viability was determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used. The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions.A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control or negative control, respectively.A relevant increase of the mutation frequency should be dose-dependent.A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration.Results of test groups are rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.See also the next field "Any other information on..."

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The summary results are attached.

No precipitation was observed by the naked eye in any of the main experiments performed.

No relevant toxic effects indicated by a relative cloning efficiency 1 or a relative total growth of less than 50 % of survival were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

The isolated but strong reduction of the relative cloning efficiency 1 observed in the second culture of the first experiment with metabolic activation (2.1 %) was not considered valid. No toxic effects at all were indicated by the relative total growth of the same culture or by any of the two parameters of toxicity of the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The threshold of 126 plus each solvent control count was not reached or exceeded at any test

point.

A significant dose dependent trend in the regression analysis of the mutation frequency was solely determined in the first culture of the second experiment (without metabolic activation). Since the mutation frequency neither exceeded the historical range of negative and solvent controls nor the threshold as indicated above, the statistical result is considered as biologically irrelevant fluctuation.

In this study the range of the negative and solvent controls was from 48 up to 131 mutant colonies per 10E6 cells; the range of the acceptable groups treated with the test item was from 42 up to 179 mutant colonies per 10E6 cells. The lowest negative value (48 colonies per 10E6 cells) fell just short of the recommended 50 – 170 x 10E6 control range. The mean value of both parallel cultures however, (55 and 48 equal to 51.5 colonies per 106 cells) is fully acceptable.

MMS (19.5 μg/mL in experiment I and 13 μg/mL in experiment II) and CPA (3.0 μg/mL) showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

The relative total growth of the positive control in experiment I, culture II with metabolic activation fell short of the 10 % limit. However, the mean value of both parallel cultures (21.4 % and 5.4 %, equal to a mean of 13.4 %) remained within the acceptable range.

Conclusions:

Interpretation of results (migrated information):negativeIt can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of DSM-ADH (adipic acid dihydrazide) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The highest applied concentration (1800 μg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for selection of genetic toxicity endpoint
Each of the 4 available studies is a key study.

Short description of key information:
Two Ames tests, one Mouse Lymphoma test and an in vitro chromosome aberration assay with human lymphocytes are available. In each test a negative result was obtained.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No indication for the need to classify adipic acid dihydrazide as a genotoxic agent was obtained.