Zinc 5-nitroisophthalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Zinc 5-nitroisophthalate has been tested in a bacterial reverse mutation assay, in a gene mutation test, and in a cytogenicity test. All tests showed a negative response, thus, Zinc 5-nitroisophthalate does not require classification for mutagenic properties. 

Endpoint Conclusion: No adverse effect observed (negative).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06/2017-

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Version: adopted 29 July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 16293305-0 (TV 20)
- Expiration date of the lot/batch: 21 July 2018


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light, dry in closed containers


OTHER SPECIFICS:
The pH value detected with the test item was within the physiological range. The solvent was compatible with the survival of the cells and the S9 activity. (Please refer to Tab.1 in the section: "any other information on materials and methods incl. tables")

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human (primary)

Details on mammalian cell type (if applicable):

CELLS USED
Human peripheral blood lymphocytes from young healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability (pre-experiment: male, age below 35 years, main experiment I: female, 32 years old, main experiment II: male, 23 years old).
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
-Complete Culture Medium: RPMI 1640 medium (supplemented with 15% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin solution, 2.4 µg/mLphytohaemagglutinin (PHA))
-Treatment Medium (short-term exposure):Complete culture medium without FBS
-After Treatment Medium / Treatment Medium (long-term exposure): Complete culture medium with 15 % FBS and 6 µg/mL cytochalasin B

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Experiment I with short-term exposure (4 h):
without metabolic activation: 2.5, 5, 10, 20, 30, 50, 100, 150, 200, 250 and 500 µg/mL
with metabolic activation: 5, 10, 30, 50, 75, 100, 150, 200, 250 and 500 µg/mL

Experiment II with long-term exposure (44 h):
without metabolic activation: 2.5, 5, 10, 20, 30, 50, 100, 150, 200, 250 and 500 µg/mL

Justification for top dose:
Precipitation visible by the inverted microscope at the beginning of the treatment was observed at concentrations of 250 µg/mL and higher with and without metabolic activation. At the end of treatment, precipitate of the test item visible by unaided eye and inverted microscope was noted at concentrations of 250 µg/mL and higher with and without metabolic activation.

Vehicle / solvent:

Due to the nature of the test item it was not possible to prepare a solution of the test item with cell culture medium (RPMI). Therefore the test item was suspended in dimethylsulfoxide (DMSO) and diluted in cell culture medium to reach a final concentration of 1% v/v DMSO in the samples.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Final concentrations: 900 and 1400 µg/mL; clastogenic control (without metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Final concentration: 15 µg/mL; clastogenic control (with metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

Final concentrations: 0.04 and 0.8 µg/mL; aneugenic control (without metabolic activation)

Details on test system and experimental conditions:

DURATION
Please refer to Tab. 2. in the field: 'Any other information on materials or methods incl. tables'.

SPINDLE INHIBITOR:
Cytochalasin B

NUMBER OF REPLICATIONS:
Duplicate cultures were performed at each concentration level except for the pre-experiment.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
At the end of the cultivation, the complete culture medium was removed. Subsequently, the cells were treated with cold hypotonic solution (0.075 M KCl) for some minutes at room temperature and immediately centrifuged. The pellet was resuspended with a solution consisted of fixation solution + NaCl 0.9% (1+1) and centrifuged. After that the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. The cells were stained with acridine orange solution.

NUMBER OF CELLS EVALUATED:
2000 (if possible)


CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
For each dose group at least 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech*, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered**.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI) was determined from 500 cells according to the following formula=(N mononucleate cells x 1) + (N binucleate cells x 2) + (N multinucleate cells x 3) / N total


*Fenech M. (2000). The in vitro micronucleus technique. Mutation Research 455, 81 – 95
**Kalweit S., D. Utesch, W. von der Hude and S. Madle (1999). Chemically induced micronucleus formation in V79 cells – comparison of three different test approaches. Mutation Research 439, 183 – 190

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined:

- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

Value comparison:
Statistical significance at the 5% level (p < 0.05) was evaluated by the non-parametric Chi-square test. The p value was used as a limit in judging for significance levels in comparison with the concurrent solvent control.

Trend test:
Statistical significance at the 5% level (p < 0.05) was evaluated by the Chi-square test for trend. The p value was used as a limit in judging for significance levels.

Species / strain:

lymphocytes: human (primary)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487*. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.

In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to the highest concentration evaluated. In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 50 µg/mL. At a concentration of 100 µg/mL a cytostasis of 40% and at a concentration of 200 µg/mL a cytostasis of 53% was noted.

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 50 µg/mL. At a concentration of 100 µg/mL a cytostasis of 35%, at a concentration of 200 µg/mL a cytostasis of 43%, at a concentration of 250 µg/mL a cytostasis of 36% and at a concentration of 500 µg/mL a cytostasis of 41% was noted.


CLASTOGENICITY/ANEUGENICITY
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.65%) was within the historical control limits of the negative control (0.28% – 1.21%) and the micronucleated cell frequency of the solvent control (1.10%) was within the historical control limits of the solvent control (0.16% – 1.84%). The mean values of micronucleated cells found after treatment with the test item were 0.55% (50 µg/mL), 0.40% (100 µg/mL) and 0.77% (200 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.05%) was within the historical control limits of the negative control (0.25% – 1.33%) and the micronucleated cell frequency of the solvent control (0.80%) was within the historical control limits of the solvent control (0.28% – 1.66%). The mean values of micronucleated cells found after treatment with the test item were 0.59% (150 µg/mL), 0.75% (200 µg/mL) and 0.51% (250 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.80%) was within the historical control limits of the negative control (0.28% – 1.21%, Table 15) and the micronucleated cell frequency of the solvent control (0.60%) was within the historical control limits of the solvent control (0.16% – 1.84%, Table 15). The mean values of micronucleated cells found after treatment with the test item were 0.65% (50 µg/mL), 0.70% (100 µg/mL), 0.60% (200 µg/mL), 0.85% (250 µg/mL) and 1.00% (500 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative and solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.

The nonparametric Chi-square Test was performed to verify the results in both experiments. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II with and without metabolic activation.
The Chi-square Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.

Ethylmethanesulfonate (EMS, 900 and 1400 µg/mL) and cyclophosphamide (CPA, 15 µg/mL) were used as clastogenic controls. Colchicine (Colc, 0.04 and 0.8 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. The values of the micronucleated cell frequency of the positive controls were within the historical control limits of the positive controls.This demonstrates the validity of the assay.

*OECD Guideline for the Testing of Chemicals, Section 4, No. 487, “In Vitro Mammalian Cell Micronucleus Test", adopted 29 July, 2016

Remarks on result:

other:

Remarks:

Please refer to 'Additional information on cytotoxicity'

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Zinc 5-nitroisophthalate did not induce structural and/or numerical chromosomal damage in human lymphocytes.
Therefore, Zinc 5-nitroisophthalate is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

- stability of the test substance throughout the study period has been verified by reanalysis.
- homogeneity of the test substance was guaranteed by mixing before preparation of the test substance formulations.
- stability of the test substance at room temperature in water over a period of 96 hours has been verified analytically.

Target gene:

TA98:his D3052
TA100 & TA1535: his G 46
TA1537: his C 3076
E. coli WP2 uvrA: trp-

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation system:

S9-mix (containing 10 % S9): MgCl2 (8 mM); KCl (33 mM); glucose-6-phosphate (5 mM); NADP (4 mM); phosphate buffer (pH 7.4)(15 mM)

Test concentrations with justification for top dose:

Experiment 1: 0, 29, 145, 725, 3625, and 7250 µg/plate (with and without metabolic activation)
Experiment 2: 0, 20, 100, 500, 2500, and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; experiment 1) & preincubation (experiment 2)

EXPERIMENT 1:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (A,es et al., 1975; Macron & Ames, 1983)*.
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.6% agar+ 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin (salmonella strains) or 0.5 mM tryptophan (E.coli strain)) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S-9 mix (in tests with metabolic activation)
or
0.5 mL phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates; Salmonella strains) or minimal agar plates (E . Coli strain)) within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants (Salmonella strains) or trp- (E.coli strain)) are counted.

EXPERIMENT 2:
The experimental procedure is based on the method described by Yahagi et al. (1977)* and Matsushima et al. (1980)*.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S-9 mix in all experiments and indicated in the tables.

TITER DETERMINATION
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10^-6)
0.5 mL S-9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

*References:
- Ames, B.N.; Mccann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mut. Res., fil_, 347 - 364 (1975)
- Maron, D.M.; Ames, B.N.: Revised method for the Salmonella mutagenicity test. Mut. Res., 113, 217 - 233 (1983)
- Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, M.: Mutagenicities of N-nitrosoamines in Salmonella. Mut. Res., 48, 121 - 130 (1977)
- Matsushima, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai, A.: Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens.
Springer Verlag Berlin, Heidelberg, N.ew York (1980).

Rationale for test conditions:

Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st experiment.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out
independently of each other.

Statistics:

Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test substance precipitation was found from about 2500 μg/plate onward.

EXPERIMENT 1 / EXPERIMENT 2
- with and without metabolic activation: no increase in the number of his+ or trp+ revertants was observed for any concentrations.

INFORMATION ON CYTOTOXICITY:
A slight decrease in the number of revertants was occasionally observed.

Please also refer for results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Attached backgrounds material" below.

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Reference 3

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:

09/2017-

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version: 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 16293305-0 (TV20)
- Expiration date of the batch: 21 July 2018
- Purity test date:


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
- Stability under test conditions: Determinations of stability and characteristics of the test article were the responsibility of the Sponsor
- Solubility of the test substance in the solvent/vehicle: Further assessments confirmed that Zinc 5-nitroisophthalate (CAS No. 60580 61 2) formed a cloudy white, free-flowing suspension in Acetone at concentrations up to approximately 10 mg/mL. The solubility limit in culture medium was in the range of 50 to 100 µg/mL, as indicated by precipitation at the higher concentration which persisted for at least 22 hours after test article addition.
In accordance with the regulatory test guidelines applicable for this study (see Section 2.4), no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparation of test article were employed.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test article formulations of Zinc 5-nitroisophthalate (CAS No. 60580 61 2) were prepared under subdued lighting in Acetone, with the aid of vortex mixing, warming at 37°C and sonication, as required, to give the maximum required concentration. Subsequent dilutions were made using Acetone. The test article solutions were protected from light and used within approximately 2 hours of initial formulation.

OTHER.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration analysed (25 µg/mL) as compared to the concurrent vehicle controls

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Methods for maintenance in cell culture if applicable: For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC

MEDIA USED
- RPMI 1640 media supplied containing L-glutamine and HEPES were prepared as shown in Tab. 1. (please refer to the field: 'Any other information on materials and methods incl. tables')
- Type and identity of media including CO2 concentration if applicable:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver S-9 used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA. Prepared from male Sprague Dawley rats induced with Aroclor. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM)

Test concentrations with justification for top dose:

- without metabolic activation: 5, 10, 12.5, 15, 17.5, 20, 25, and 50 µg/mL
- with metabolic activation: 2.5, 5, 10, 15, 20, 25, and 50 µg/mL

JUSTIFICATION FOR TOP DOSE
Tested up to the limit of solubility in culture medium, for 3 hours in the absence and presence of a rat liver metabolic activation system (S 9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone (further diluted in Culture medium (RPMI)
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that Zinc 5-nitroisophthalate (CAS No. 60580 61 2) was insoluble in anhydrous analytical grade dimethyl sulphoxide (DMSO), dimethylformamide (DMF), tetrahydrafuran (THF) and ethanol. Further assessments confirmed that Zinc 5-nitroisophthalate (CAS No. 60580 61 2) formed a cloudy white, free-flowing suspension in Acetone at concentrations up to approximately 10 mg/mL. The solubility limit in culture medium was in the range of 50 to 100 µg/mL, as indicated by precipitation at the higher concentration which persisted for at least 22 hours after test article addition.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle Acetone diluted 100-fold in the treatment medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Final concentration: 0.15 and 0.20 µg/ml; used in absence of a metabolic activation system; prepared in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle Acetone diluted 100-fold in the treatment medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Final concentration: 2.0 and 3.0 µg/ml; used in presence of a metabolic activation system; prepared in DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): Mutation experiment: 384 wells at 2 x 10^4 cells/well

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 to 14 days

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS:
Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments).


DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, adjusted relative survival rate (adjusted %RS)

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:

1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 experiments in the historical negative control database (mean MF +/ 2 standard deviations.

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines*. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

*Robinson W D, Green M H L, Cole J, Garner R C, Healy M J R and Gatehouse D (1990). Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed D J Kirkland) Cambridge University Press, pp 102-140

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Please refer to 'Additional information on results'

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

MUTATION EXPERIMENT
Following 3 hour treatment in the absence and presence of S-9, no statistically significant increases in MF, compared to the vehicle control, were observed following treatment with Zinc 5-nitroisophthalate (CAS No. 60580 61 2) at any concentration analysed. All concentrations in the absence of S-9 were within the range generated by the last twenty experiments performed in this laboratory (1.29 to 7.63 mutants per 106 viable cells). In the presence of S-9 the MF of a single, intermediate concentration (5 µg/mL) was outside the range generated by the last twenty experiments (2.43 to 6.48 mutants per 106 viable cells). In addition, a statistically significant (p≤0.05) linear trend was observed in the absence of S-9. However, in the absence of any statistically significant increases in MF for any treated culture these observations are considered not biologically relevant.

CYTOTOXICITY
In the Mutation Experiment ten concentrations were tested, ranging from 1.25 to 50 µg/mL in the absence of S-9 and from 0.625 to 100 µg/mL in the presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations tested (17.5 to 50 µg/mL in the absence of S-9 and 20 to 100 µg/mL in the presence of S-9). Following the 3 hour treatment incubation period, precipitate was observed at the highest concentration tested in the absence of S-9 (50 µg/mL) and the highest two concentrations tested in the presence of S-9 (50 and 100 µg/mL). The absence of precipitate in the highest non-precipitating concentrations (25 µg/mL in the absence and presence of S-9) was confirmed by haemocytometer. The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and higher concentrations were discarded. All concentrations were retained in the absence of S-9. Seven days after treatment, the lowest two concentrations in the absence and presence of S-9 (1.25 to 2.5 µg/mL and 0.625 to 1.25 µg/mL, respectively) were not selected as there were sufficient non-toxic concentrations to determine an appropriate toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentration analysed was 50 µg/mL in the absence and presence of S-9 (limited by the appearance of post treatment precipitate), which gave 22% and 32% RS, respectively (see following table).

CYTOTOXICITY RANGE-FINDER
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 3.125 to 100 µg/mL (the maximum practicable concentration). The test article, at the highest concentration tested, was a free flowing, doseable suspension and therefore all concentrations cited in this report are considered nominal. Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations tested in the absence and presence of S-9 (25 to 100 µg/mL). As the test article, at the highest concentration tested, was a free flowing suspension it was important to confirm that no micro precipitate/undissolved test article was present in the cultures post treatment. The absence of such precipitate in the highest non precipitating concentration observed by eye (12.5 µg/mL in the absence and presence of S-9) was confirmed by haemocytometer. The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentrations to give 10% relative survival (RS) were 12.5 µg/mL in the absence of S-9 (limited by toxicity) and 25 µg/mL in the presence of S-9 (limited by the appearance of post treatment precipitate), which gave 47% and 97% RS, respectively:

Remarks on result:

other: Please refer to 'Additional information on results'

Conclusions:

It is concluded that Zinc 5-nitroisophthalate (CAS No. 60580 61 2) did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to the limit of solubility in culture medium, for 3 hours in the absence and presence of a rat liver metabolic activation system (S 9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity – in vitro results

Gene mutation in bacterial test systems

Engelhardt and Hoffmann (1999, GLP) evaluated the mutagenic potential of Zinc 5-nitroisophthalate in a bacterial reverse mutation assay. In two different experiments, four Salmonella strains(TA98, TA100, TA1535, and TA1537) and one E.coli strain (WP2uvrA) were tested either in a plate incorporation experiment (29-7250 µg/plate) or in a preincubation experiment (20-5000 µg/plate). Both experiments were performed either with or without metabolic activation. After exposure to the test item for 48‑72 hours, the plates were scored for revertant colony number. None of the doses applied resulted in an increase in revertant colony number under any condition tested. According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

 

Cytogenicity in mammalian cells

In a GLP study (Donath, 2018), cytogenic activity of Zinc 5-nitroisophthalate was assessed via a micronucleus test. Human peripheral blood lymphocytes were exposed to the test item without a metabolic activation system for either 4 or 44 hours at concentrations of 2.5-500 µg/mL. Additionally, the cells were also tested in presence of a metabolic activation system (S-9 from activated rat liver). Whenever possible, 2000 cells were scored for micronuclei occurrence. During the study described and under the experimental conditions reported, the test item Zinc 5-nitroisophthalate did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, Zinc 5-nitroisophthalate is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

 

Gene mutation in mammalian cells

A GLP study (Hargreaves, 2018) evaluated Zinc 5-nitroisophthalate for the potential to induce mutations at the hprt locus in mouse lymphoma L5178Y cells. To this end, cells were exposed to the test item either without metabolic activation or in combination with a metabolic activation system (S-9 from activated rat liver). In both cases the top dose was 50 µg/mL based on solubility limits. The cells were treated for three hours. After a total of 13-14 days the mutant cell number was counted, and the mutant frequency was determined subsequently. It is concluded that Zinc 5-nitroisophthalate did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to the limit of solubility in culture medium, for 3 hours in the absence and presence of a rat liver metabolic activation system (S 9).

Justification for classification or non-classification

Genetic toxicity, in vitro

All studies present are GLP compliant and showed not any effect in a bacterial reverse mutation assay, in a mammalian cell gene mutation test (hprt) or in a mammalian cell micronucleus test. The classification criteria according to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no classification applicable.