Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Version: adopted 29 July, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Molecular weight: 274.5 g/mol
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")

Method

Target gene:
not applicable
Species / strain
Species / strain:
lymphocytes: human (primary)
Details on mammalian cell lines (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 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:
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.
Controlsopen allclose all
Negative controls:
yes
Solvent 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)
Negative controls:
yes
Solvent 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)
Negative controls:
yes
Solvent 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 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.

Results and discussion

Test results
Species / strain:
lymphocytes: human (primary)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Please refer to 'Additional information on cytotoxicity'
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

Applicant's summary and conclusion

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.