1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione, zinc salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity: Negative; OECD 471, SafePharm (2002)

Chromosome Aberration: Negative; OECD 473; SafePharm (2004)

Mammalian Cell Gene Mutation: Negative; OECD 476; CCR (2012) - read-across from 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2)

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:

September - October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/Beta-naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Preliminary Toxicity Study:
The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 500, 1500 and 5000 microg/plate.
Mutation Study 1 and 2:
Five concentrations of the test material (50, 150, 500, 1500 and 5000 migrog/plate) were assayed.

Vehicle / solvent:

Dimethyl sulphoxide was selected as the vehicle of choice because it formed a good partial solution/suspension with the test material at 50mg/ml.

Negative solvent / vehicle controls:

yes

Remarks:

A solvent treatment group was used as vehicle control

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 3 microg/plate for TA100 and 5 microg/plate for TA1535

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 microg/plate for TA1537

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 0.5 microg/plate for TA102

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 microg/plate for TA98

Positive control substance:

other: 2-Aminoanthracene 1 microg/plate for TA100 and 2 microg/plate for TA 1535 and TA 1537

Positive control substance:

benzo(a)pyrene

Remarks:

Used in the S9 series of plates

Migrated to IUCLID6: 5 microg/plate for TA98

Positive control substance:

other: 1,8-Dihydroxyanthraquinone 10 microg/plarte for TA102

Remarks:

Used in the S9 series of plates

Details on test system and experimental conditions:

Preliminary Toxicity Study:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate. The test was performed by mixing 0.1ml of bacterial culture (TA100), 2ml of molten, trace histidine supplemented, top agar, 0.1ml of test material fomulation, 0.5ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Volgel-Bonner Minimal agar (30 ml/plate). Ten doses of the test material and a vehicle control (dimethyl suplhoxide) were tested. In addition, 0.1ml of the maximum concentration of the test material and 2ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 deg C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Study - Experiment 1:
Five concentrations of the test material 50, 150, 500, 1500 and 5000 microg/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All the plates were incubated at 37 degC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2:
The second experiment was performed using methodology as described in Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9E9 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strain to mutagenic exposure and the integrity of the S9-mix.

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level, although a decrease in the frequency of revertant colonies was noted at 5000 microg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 microg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence of absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

ZMB2 is considered to be non-mutagenic under the conditions of this test.

Reference 2

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according OECD 476

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The study was performed to investigate the potential of 1,3-Dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first and second experiment with and without metabolic activation.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: The cells have a stable karyotype with a modal chromosome number of 22

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

exposure S9 concentrations
period mix in µg/mL
Experiment I
4 hours - 31.3 62.5 125.0 250.0 500.0 750.0 P 1000.0 P
4 hours + 15.6 31.3 62.5 125.0 250.0 500.0 P 1000.0 P
Experiment II
4 hours - 31.3 62.5 125.0 250.0 P 375.0 P 500.0 P 750.0 P
4 hours + 15.6 31.3 62.5 125.0 P 250.0 P 500.0 P 1000.0 P
p = precipitation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS; ethylmethane sulfonate; With metabolic activation: DMBA; 7,12-dimethylbenz(a)anthracene

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Relevant cytotoxic effects were noted at 250.0 µg/mL and above without metabolic activation. In the presence of metabolic activation cytotoxic effects as described above were not observed up to the maximum concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported ZMB2 did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, ZMB2 is considered negative in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according OECD 476

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to Section 13.2 for read-across justification document.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Relevant cytotoxic effects were noted at 250.0 µg/mL and above without metabolic activation. In the presence of metabolic activation cytotoxic effects as described above were not observed up to the maximum concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported ZMB2 did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, ZMB2 is considered negative in this HPRT assay.

Executive summary:

In a one-to-one read-across approach, the substance 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (source substance) is considered appropriate for direct read-across (one-to-one) to zinc 4-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide 7-methyl-2-thioxo-2,3-dihydrobenzimidazol-1-ide (target substance) for the endpoint genetic toxicity in vitro. In conclusion, under the experimental conditions reported the substance did not induce gene mutations at the HPRT locus in V79 cells. A full justification for the read-across approach is presented in IUCLID Section 13.2.

Reference 4

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:

11 May 2004 - 16 November 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to GLP and OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

For each experiment sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study were determined usign BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculated the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in house" with L-gutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37 degC with 5% CO2 in air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutin (PHA) at 90 microg/ml final concentration.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Without S9: 31.25, 62.5, 125, 250, 375, 500 microg/ml
With S9: 31.25, 62.5, 125, 250, 375, 500 microg/ml

Vehicle / solvent:

Dimethyl sulphoxide (DMSO).

Negative solvent / vehicle controls:

yes

Remarks:

Parallel with test material

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Migrated to IUCLID6: 0.4 and 0.2 microg/ml

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Migrated to IUCLID6: 5.0 microg/ml

Details on test system and experimental conditions:

The test material was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared. The molecular weight of the test material was 393.9 and the maximum dose level in the preliminary toxicity test would normally have been the 10mM maximum recommended dose level of 3940 microg/ml. However, the maximum dose level was limited to 2000 microg/ml because the test material precipitate was observed to aggregate into a single mass at and above 1970 microg/ml and, therefore, effectively reduce its maximum exposure to the cells. The maximum dose levels selected for the main experiments were further reduced due to test material toxicity. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.

Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispersed into sterile plastic flasks for each culture:

9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

With Metabolic Activation (S9) Treatment:
After approximately 48 hours incubation at 37 degC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 ml of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 ml of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 ml of the appropriate solution was added to the cultures. 1 ml of 20% S9 mix (ie 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Tes of Experiment 1.

In Experiment 2, 1 ml of 10% S9 mix (ie 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 ml.

After 4 hours at 37 degC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37 degC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment:
In Experiment 1, after approximately 48 hours incubation at 37 degC with 5% CO2 in humidified air in cultures were decanted into tubes and centrifuged. Approximately 9 ml of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 ml of the appropriate vehicle control, test material solution or positive control solution. The total volume for each culture was a nominal 10 ml.

After 4 hours at 37 degC, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 ml. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 ml of vehicle control, test material dose solution or positive control solution. The nominal final volume of each culture was 10 ml. The cultures were then incubated at 37 degC for 24 hours.

Preliminary Toxicity Test:
A preliminary toxicity test was performed on cell cultures using a 4 hours exposure time with and without metabolic activation followed by a 20 hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 15.6 to 2000 microg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main study.

- Experiment 1:
i) 4 hour exposure to the test material without S9 mix followed by 20 hour culture in treatment-free media prior to cell harvest.

ii) 4-hour exposure to the test material with S9 mix followed by 20 hour culture in treatment free media prior to cell harvest.

- Experiment 2:
i) 24 hour continuous exposure to the test material without S9 mix prior to cell harvest.

ii) 4 hour exposure to the test material with S9 mix followed by 20 hour culture in treatment free media prior to cell harvest.

Cell Harvest:
Mitosis was arrested by addition of demecolcine two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KCl. After approximately fourteen minutes including centrifugation, most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was charged at least three times and the cells stored at 4 degC for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads:
The lymphocytes were resuspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and cover slipped using mounting medium.


The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Evaluation criteria:

Where possible the first 100 consecutive well spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, the concurrent vehicle value using Fisher's Exact test.

Species / strain:

human lymphoblastoid cells (TK6)

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:

Toxicity Test:
The dose range for the Preliminary Toxicity Test was 15.6 to 2000 microg/ml. The maximum dose was based on the lowest acceptable precipitating dose level. A precipitate of the test material was observed in the parallel blood free cultures at the end of the exposure, at and above 250 micog/ml, in the 4(2) hour pulse exposure groups and at and above 125 microg/ml in the continuous exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 250 microg/ml in all three of the treatment groups. The test material showed no clear evidence of toxicity, as indicated by dose related reductions in mitotic index in the surviving dose levels, in any of the exposure groups.

Chromosome Aberration Test Experiment 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were scorable metaphases present up to 375 microg/ml in the absence and presence of metabolic activation (S9).
The mitotic index data confirms the qualitative observations in that a dose related inhibition of mitotic index was observed, and that 65% mitotic inhibition was achieved at 375 microg/ml in the presence of S9. In absence of S9 the dose relationship was not as clear cut, and the maximum mitotic inhibition of 41% was achieved at 375 microg/ml.
The maximum dose level selected for metaphase analysis, 375 microg/ml, was based on toxicity in both the absence and presence of S9.
All the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases int he frequency of cells with aberrations either in the absence or presence of metabolic activation. It should be noted that in presence of S9 at 375 microg/ml it was only possible to score 36 metaphase cells for the A culture. This was due to the level of test material toxicity observed in this culture. However, with no evidence of a response being observed in this experiment and in Experiment 2, it was, therefore, considered that the overall result and integrity of the study was not affected.
The test material did not induce any statistically significant increases in the numbers of polyploidy cells at any dose level in either of the exposure groups.

Chromosom Aberration Test - Experiment 2:
The qualitative assessment of the slides determined that there were scorable metaphases present up to 125 microg/ml in the absence of S9. In the presence of S9 the maximum test material dose level with scorable metaphases was 375 microg/ml.
The mitotic index data confirm the qualitative observations in that a dose related inhibition of mitotic index was observed, and that 39% mitotic inhibition was achieved at 125 microg/ml in the absence of S9. In the presence of S9 52% mitotic inhibition was achieved at 375 microg/ml.
The maximum dose levels selected for metaphase analysis, 125 and 375 microg/ml in the absence and presence of S9 respectively were based on toxicity.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected tange. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

Interpretation of results (migrated information):
negative

ZMB2 did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolizing system in either of two separate experiments. ZMB2 is therefore considered to be non-lactogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Not applicable, no adverse effects were observed in the required studies.

Additional information

In vitro genotoxicity studies were conducted on the registered substance 2H-Benzimidazole-2-thione, 1,3-dihydro-4(or 5)-methyl-, zinc salt (2:1) (ZMB2). Both the AMES test (OECD 471) and chromosome aberration test (OECD 473) produced negative results for mutagenicity. In addition, a mammalian cell gene mutation assay (OECD 476) was read-across from 1,3-dihydro-4(or 5)-methyl-2H-benzimidazole-2-thione (MB2) in a one-to-one read-across approach, this study was also negative. In conclusion, the required studies for registration of this substance are submitted and conclude that the substance is not mutagenic. Read-across from ZMB2 to MB2 is justified fully in Section 13.2 of IUCLID (LANXESS, 2018). In summary, ZMB2 exhibits its salt characteristic in aqueous solution, i.e., dissolving in water to MB2 and zinc ions (Zn(OH)2) in a matter of seconds.

In support of the endpoint conclusion, it is also worth noting that the zinc hydroxide (ZN(OH)2, EC# 243 -814 -3) which is part of the inorganic zinc category under REACH, is also not considered mutagenic. Thus, the inorganic zinc produced following hydrolytic dissociation will not influence the conclusion of non-mutagenic for this substance. A further update to this dossier is planned in the near future, where the relevant zinc data will be presented in support of this conclusion.

Justification for classification or non-classification

In an Ames, Chromosome Aberration, and appropriate read across to MB2 (CAS Number 53988-10-6) CHO study, ZMB2 is considered to be negative.

In accordance with Regulation No 1272/2008 Table 3.5.1 ZMB2 is not be classified for genetic toxicity.