Zinc di(benzothiazol-2-yl) disulphide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

ZMBT is composed of two 2-Mercaptobenzothiazole (MBT) molecules (CAS 149-30-4, EC 205-736-8) associated with zinc ion (mass content ZMBT: 84% MBT, 16% Zn2+). It was shown in hydrolysis studies that under acidic conditions (pH 3) a rapid degradation of ZMBT to MBT and zinc ions Zn2+ was observed. A read-across with toxicological data for MBT as source is thus considered adequate. For justification of the read-across see separate Read-Across Justification Document attached to the IUCLID.

ZMBT was tested in a bacterial reverse mutation test in accordance to OECD test guideline 471 and in compliance with GLP. The treatments were performed up to the maximum recommended concentration (5000 µg/plate) with and without S9 according to current regulatory guidelines. ZMBT did not induce mutations in the Ames test when tested under the conditions of this study (Covance 2020).

An in vitro HPRT mammalian cell gene mutation test was performed with ZMBT in mouse lymphoma cells. The test was conducted according to OECD test guideline 476 and in compliance with GLP. Based on range-finder results showing cytotoxicity the Mutation Experiment was conducted with ten concentrations of up to 50.00 μg/mL in the absence of S9 and up to 100.0 μg/mL in the presence of S9. Test solutions were formulated in anhydrous analytical grade DMSO. It is concluded that ZMBT did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations, in the absence and presence of a rat liver metabolic activation system (S9). (Covance 2020)

An in vitro micronucleus assay test was performed to evaluate the clastogenic and aneugenic potential of ZMBT according to OECD test guideline 487 and in compliance with GLP. The test article was formulated in anhydrous analytical grade DMSO. The highest concentrations tested in the Micronucleus Experiment were either limited by solubility or by toxicity. Treatment of cells resulted in frequencies of micronucleated binucleate (MNBN) cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls in cultures with and without S9 mix at the highest two concentrations analysed after 3 hours treatment and in cultures without S9 after 24 hours treatment. The increases observed exceeded the normal range in both replicate cultures for both concentrations. A statistically significant linear trend was observed in all treatments, indicating a positive results. It is concluded that ZMBT did induce micronuclei in cultured human peripheral blood lymphocytes (Covance 2020).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 October 2019 - 13 November 2019

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL
- Identification: ZMBT; Zinc di(benzothiazol-2-yl) disulphide
- Chemical Name: Zinc di(benzothiazol-2-yl) disulphide
- Common Name: ZMBTCAS Number155-04-4
- Lot / Batch Number: LAB20190801
- Expiry Date: June 2021
- Appearance: Light yellow powder
- Purity: 99.0%
- Correction Factor: Not required
- Molecular Weight: 397.89

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Experiment 1: μg/plate: 5, 16, 50, 160, 500, 1600 and 5000
Experiment 2: μg/plate: 156.25, 312.5, 625, 1250, 2500 and 5000
Justification for top dose: the maximum recommended concentration according to current regulatory guidelines

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 0.5% methyl cellulose

- Justification for choice of solvent/vehicle: test item was not soluble in water or assay suitable organic solvents at appropriate concentrations. Therefore, the study was performed using formulations prepared in 0.5% methyl cellulose. Formulation assessment confirmed that the test article did form a suitable homogenous suspension in 0.5% methyl cellulose (0.5% MC) at 50 mg/mL.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

PREPARATION
Test article stock solutions were prepared by formulating ZMBT; Zinc di(benzothiazol-2-yl) disulphide under subdued lighting in 0.5% MC with the aid of Silverson mixing, vortex mixing, and warming at 37°C (where required), to give the maximum required treatment concentration. Subsequent dilutions were made using 0.5% MC. All suspensions were homogenized by inversion prior to dilution or treatment. The test article solutions were protected from light and used within approximately 4 hours of initial formulation.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^8 to 10^9 cells/mL
- Test substance added in medium: Platings were achieved by the following sequence of additions to 2 mL supplemented molten top agar at 45±1°C:0.1 mL bacterial culture, 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control, 0.5 mL 10% S-9 mix or buffer solution followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37°C. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted.

As the results of Experiment 1 were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and placed in an orbital incubator set to 37°C for 20 minutes, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
It may be noted that initial Experiment 2 treatments of strains TA100, TA1535 and TA1537 in the presence of S-9 were invalidated due to failure of the positive controls, and the data are not reported. These Experiment 2 treatments in the presence of S-9 were repeated in order to provide the data for these strains which are presented in this report.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1.A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2.The positive trend/effects described above were reproducible.

The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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:

STUDY RESULTS
Ames test:
Mutation Experiment 1: Following these treatments, evidence of toxicity in the form of a reduction in the number of revertants was observed at 5000 μg/plate in strain TA102 in the absence and presence of S-9.
Mutation Experiment 2: Following these treatments, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 5000 μg/plate in strains TA98 and TA102 in the presence of S-9 and at 2500 μg/plate and above in strain TA102 in the absence of S-9.

As the test article was treated as a suspension, any observations regarding the presence or otherwise of particulate test article (precipitate) in the assay system are not considered relevant, and are therefore not reported.

Data Acceptability and Validity
The vehicle control counts fell within the laboratory’s historical ranges, with the exception of one vehicle control count for strain TA98 in the absence of S-9 (Experiment 1) and one vehicle control count for strain TA100 in the presence of S-9 (Experiment 2) that fell slightly outside the laboratory control range. In each case, the count was comparable to the other vehicle control replicate counts and the laboratory historical control range, and therefore these data were accepted as characteristic and valid.
The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation
Following ZMBT; Zinc di(benzothiazol-2-yl) disulphide treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any ZMBT; Zinc di(benzothiazol-2-yl) disulphide mutagenic activity in this assay system.

Conclusions:

It was concluded that ZMBT; Zinc di(benzothiazol-2-yl) disulphide did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

A Bacterial reverse mutation test was conducted (Covance Laboratories Ltd, Study 8417383, 2020) to examine the potential for ZMBT to cause gene mutation. The study was conducted in accordance to OECD test guideline 471 and in compliance with GLP.

Five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation (S9) were exposed ZMBT in two separate experiments in triplicate in 0.5% methyl cellulose as vehicle. The seven concentrations of ZMBT were 5, 16, 50, 160, 500, 1600 and 5000 μg/plate in the Experiment 1, and 156.25, 312.5, 625, 1250, 2500 and 5000 μg/plate in the Experiment 2 (with pre-incubation). The treatments were performed up to this maximum recommended concentration according to current regulatory guidelines.

In Mutation Experiment 1, evidence of toxicity in the form of a reduction in the number of revertants was observed at 5000 μg/plate in strain TA102 in the absence and presence of S-9. In Mutation Experiment 2, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 5000 μg/plate in strains TA98 and TA102 in the presence of S-9 and at 2500 μg/plate and above in strain TA102 in the absence of S-9.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

It was concluded that ZMBT; Zinc di(benzothiazol-2-yl) disulphide did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate in the absence and in the presence of a rat liver metabolic activation system (S9).

 

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January 2020 to 07 April 2020

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 using the Hprt and xprt genes)

Version / remarks:

2016

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TEST MATERIAL
- Identification: ZMBT; Zinc di(benzothiazol-2-yl) disulphide
- Chemical Name: Zinc di(benzothiazol-2-yl) disulphide
- Common Name: ZMBT
- CAS Number: 155-04-4
- Lot / Batch Number: LAB20190801
- Expiry Date: June 2021
- Appearance: Light yellow powder
- Purity: 99.0%
- Correction: Factor Not required
- Molecular Weight: 397.89

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

tk+/- (3.7.2C)

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Test concentrations with justification for top dose:

Range-Finder (with and without S9) (μg/mL): 3.125, 6.25, 12.50, 25.00, 50.00 and 100.0
Mutation Experiment (without S9) (μg/mL): 2.50, 5.00, 10.00, 20.00, 25.00, 30.00, 35.00, 40.00, 45.00 and 50.00
Mutation Experiment (with S9) (μg/mL): 20.00, 30.00, 40.00, 45.00, 50.00, 55.00, 60.00, 70.00, 80.00 and 100.0
Justification for top dose: solubility of 10.00 mg/mL was maximum solubility that could be achieved (i.e. 100 µg/mL in the mutation experiment).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: the test item was not readily soluble in the standard solvents suitable for this assay.

Preliminary solubility data indicated that ZMBT; Zinc di(benzothiazol-2-yl) disulphide was not readily soluble in the standard solvents suitable for this assay. The maximum solubility was achieved in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to approximately 10 mg/mL, with the aid of warming at 50℃ and ultrasonication for approximately 90 minutes. The solubility limit in culture medium was in excess of 100 μg/mL, as indicated by a lack of any visible precipitation at this concentration over a period of approximately 3 hours after test article addition. A maximum concentration of 100 μg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to the maximum achievable concentration (OECD 476, 2016). Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Negative solvent / vehicle controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

PREPARATION:
Test article stock solutions were prepared by formulating ZMBT; Zinc di(benzothiazol-2-yl) disulphide under subdued lighting in DMSO, with the aid of vortex mixing, warming at 50℃ and ultrasonication for approximately 90 minutes, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3 hours of initial formulation.

TREATMENT:
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.

Plating for Survival
Following adjustment of the cultures to 2 x 10^5 cells/mL after treatment, samples from these were diluted to 8 cells/mL
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator set to 37ºC and 5% (v/v) CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period
Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance:
without S9: 0, 5.000, 10.00, 20.00, 25.00, 30.00 and 35.00 μg/mL
with S9: 0, 20.00, 30.00, 40.00, 45.00, 50.00, 55.00 and 60.00 μg/mL

Plating for Viability
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified set to 37ºC and 5% (v/v) CO2 in air until scoreable (8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG Resistance
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were placed in a humidified incubator set to 37ºC and 5% (v/v) CO2 in air until scoreable (12 days) and wells containing clones were identified as above and counted.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The mutant frequency (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 studies 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 (Robinson et al., 1990). 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

tk+/- (3.7.2C)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

when tested up to toxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At doses >= 35 μg/mL without S9 and >= 60 μg/mL with S-9.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity
No precipitation was observed in the absence or presence of S-9, either at the time of treatment or at the end of the treatment incubation period. Seven days after treatment, the highest three concentrations in the absence of S-9 (40 to 50 μg/mL) and presence of S-9 (70 to 100 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance.
In addition the lowest concentration in the absence of S-9 (2.5 μg/mL) was not selected as there were sufficient non-toxic concentrations.
All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 35 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9, which gave 18% and 17% RS (relative survival), respectively.

Mutation
Following 3 hour treatment in the absence and presence of S-9 the maximum concentrations analysed for viability and 6TG resistance were limited by toxicity to 35 μg/mL in the absence of S-9 and 60 μg/mL in the presence of S-9. At these concentrations RS was reduced to 18% and 17% in the absence and presence of S-9, respectively. No statistically significant increases in MF, compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends, indicating a negative result.

Formulations Analysis
Results of formulation analyses demonstrated homogeneity and stability of the samples (6 hours at room temperature) and achieved concentrations within 100±10% of the nominal test article concentrations with an RSD ≤5%, and were therefore considered acceptable.

Toxicity

Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration μg/mL	%RS	MF §	Concentration μg/mL	%RS	MF §
0	100	4.24	0	100	5.26
5	93	4.77NS	20	63	5.25NS
10	66	3.68NS	30	47	3.02NS
20	36	4.29NS	40	39	2.02NS
25	21	1.31NS	45	32	2.43NS
30	27	5.05NS	50	24	1.11NS
35	18	2.26NS	55	20	3.35NS
			60	17	1.33NS
NQO 0.15	60	23.48	B[a]P 2	67	12.34
NQO 0.2	55	25.75	B[a]P2 3	68	12.89

Test for Linear Trend
	without S9	with S9
Slope	-7.02E-08	-6.77E-08
Variance	9.09E-16	3.29E-16
b² / Sb	5.42	13.926

Absence and presence of S-9: Not Significant (negative slopes)

§: 6-TG resistant mutants/10^6 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

Conclusions:

It is concluded that ZMBT; Zinc di(benzothiazol-2-yl) disulphide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations, in the absence and presence of a rat liver metabolic activation system (S9).

Executive summary:

In vitro mammalian cell gene mutation test (Covance Laboratories Ltd., Study 8417385, 2020) was performed to access the ability of ZMBT to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The test was conducted according to EU Method B.17, OECD test guideline 476, EPA test guideline OPPTS 870.5300, and in compliance with GLP.

 

Based on range-finder results, the Mutation Experiment was conducted with ten concentrations: 2.500, 5.000, 10.00, 20.00, 25.00, 30.00, 35.00, 40.00, 45.00 and 50.00 μg/mL in the absence of S9 and 20.00, 30.00, 40.00, 45.00, 50.00, 55.00, 60.00, 70.00, 80.00 and 100.0μg/mL in the presence of S9, test solution were formulated in anhydrous analytical grade DMSO.

 

Following 3 hour treatment no statistically significant increases in mutant frequency (MF), compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends, indicating a negative result. Seven days after treatment, the highest concentrations analysed were 35 μg/mL in the absence of S9 and 60 μg/mL in the presence of S-9, which gave 18% and 17% RS, respectively.

 

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S9) and benzo(a)pyrene (B[a]P) (with S9). Therefore the study was accepted as valid.

 

It is concluded that ZMBT; Zinc di(benzothiazol-2-yl) disulphide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations, in the absence and presence of a rat liver metabolic activation system (S9).

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 February 2020 to 04 June 2020

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:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: ZMBT; Zinc di(benzothiazol-2-yl) disulphide
- Lot / Batch Number: LAB20190801
- Expiry Date: June 2021
- Appearance: Light yellow powder
- Purity: 99.0%
- Correction Factor: Not required
- Molecular Weight: 397.89

Species / strain / cell type:

lymphocytes:

Remarks:

human

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: two healthy, non-smoking female, aged 30.

- Whether whole blood or separated lymphocytes were used:Whole blood

- Whether blood from different donors were pooled or not: Blood was stored refrigerated and pooled using equal volumes from each donor prior to use

- Mitogen used for lymphocytes: yes

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively.

- concentration or volume of S9 mix and S9 in the final culture medium: 1% (v/v).

Test concentrations with justification for top dose:

Range-Finder (µg/mL): 0, 0.3628, 0.6047, 1.008, 1.680, 2.799, 4.666, 7.776, 12.96, 21.60, 36.00, 60.00, 100.0 .
Micronucleus Experiment (µg/mL): 10.00, 20.00, 25.00, 30.00, 35.00, 40.00, 42.50, 45.00, 47.50, 50.00, 55.00, 60.00.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate

TREATMENT AND HARVEST SCHEDULE:
Harvesting
Cultures were centrifuged at approx. 300g for 10 min., the supernatant removed and discarded and cells re-suspended in 4 mL (hypotonic) 0.075 M KCl at approx. 37°C for 4 min. to allow cell swelling to occur. Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation (approx. 300g, 10 min.) and re-suspension. This procedure was repeated as necessary (centrifuging at approx. 1250g, 2-3 min.) until the cell pellets were clean.

Slide Preparation
Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 h. to ensure that cells were adequately fixed. Cells were centrifuged (approx. 1250g, 2-3 min.) and re-suspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, air dried microscope slides. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 min. and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

Selection of Concentrations for the Micronucleus Experiment
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
RI, which indicates the relative number of nuclei compared to vehicle controls, was determined as follows:
RI = (number binucleate cells + 2 (number multinucleate cells))/ total number of cells in treated cultures

Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures/ RI of vehicle controls) x100

Cytotoxicity (%) is expressed as (100 – Relative RI).
Cytotoxicity was assessed from enough treatment concentrations to determine whether chemically induced cell cycle delay had occurred. A suitable range of concentrations was selected for the Micronucleus Experiment based on these toxicity data.

Selection of Concentrations for Micronucleus Analysis (Micronucleus Experiment Only)
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred.
The highest concentration selected for micronucleus analysis following 3+21 hour treatment conditions in the presence of S-9 was the highest concentration tested 100 µg/mL.
The highest concentration selected for micronucleus analysis following 3+21 hour and 24+24 hour treatment conditions in the absence of S-9 was one at which 50-60% cytotoxicity was achieved.
Slides from the highest selected concentration and two lower concentrations were taken for microscopic analysis.
The positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

Slide Analysis
Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.

A micronucleus was only recorded if:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
All slides for analysis were coded by an individual not connected with the scoring of the slides, such that analysis was conducted under blind conditions.
One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei on each slide was recorded.
Nucleoplasmic bridges (NPBs) between nuclei in binucleate cells were recorded during micronucleus analysis to provide an indication of chromosome rearrangement.
Micronucleus analysis was not conducted on slides generated from the Range-Finder treatments.

Acceptance Criteria
The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the 95th percentile of the current observed historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of cells with micronuclei. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The maximum concentration analysed under each treatment condition met the criteria specified in Section Selection of Concentrations for Micronucleus Analysis.


Validity of Study
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures
2. The frequency of MNBN cells in vehicle controls fell within the normal ranges
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range.
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest.
5. The maximum concentration analysed under each treatment condition met the criteria specified in Section Selection of Concentrations for Micronucleus Analysis.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis.

Statistics:

After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Analysis of Data

Treatment of cells with ZMBT for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (47.5 and 55 µg/mL, giving 18% and 47% reduction in RI, respectively). The increases observed exceeded the normal range in a single culture at 47.5 µg/mL and in both replicate cultures at 55 µg/mL. A statistically significant linear trend was observed, indicating a positive result for this timepoint.

Treatment of cells with ZMBT for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (80 and 100 µg/mL, giving 15% and 30% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations. A statistically significant linear trend was observed, indicating a positive result for this timepoint.

Treatment of cells with ZMBT for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (50 and 55 µg/mL, giving 24% and 42% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations. A statistically significant linear trend was observed, indicating a positive result for this timepoint.

No test article related increases in cells with NPBs were observed (data not reported).

Data for 3+21 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity
(µg/mL)							Based on RI (%)
Vehicle	A	98	349	53	500	0.91
	B	97	414	49	560	0.91
	C	79	365	56	500	0.95
	D	85	374	41	500	0.91
	Total	359	1502	199	2060	0.92	-
10	A	142	485	34	661	0.84
	B	85	381	41	507	0.91
	Total	227	866	75	1168	0.87	6
20	A	91	381	45	517	0.91
	B	106	423	65	594	0.93
	Total	197	804	110	1111	0.92	0
25	A	118	385	53	556	0.88
	B	104	353	43	500	0.88
	Total	222	738	96	1056	0.88	5
30	A	89	368	43	500	0.91
	B	115	475	70	660	0.93
	Total	204	843	113	1160	0.92	0
35	A	107	403	40	550	0.88
	B	95	387	37	519	0.89
	Total	202	790	77	1069	0.88	4
40	A	69	367	64	500	0.99
	B	91	362	47	500	0.91
	Total	160	729	111	1000	0.95	0 #
42.5	A	73	384	43	500	0.94
	B	109	435	37	581	0.88
	Total	182	819	80	1081	0.91	2
45	A	96	365	39	500	0.89
	B	100	360	40	500	0.88
	Total	196	725	79	1000	0.88	4
47.5	A	162	342	31	535	0.76
	B	154	315	31	500	0.75
	Total	316	657	62	1035	0.75	18 #
50	A	135	338	27	500	0.78
	B	169	320	29	518	0.73
	Total	304	658	56	1018	0.76	18
55	A	271	225	18	514	0.51
	B	279	203	18	500	0.48
	Total	550	428	36	1014	0.49	47 #
60	A	415	83	5	503	0.18
	B	453	42	5	500	0.1
	Total	868	125	10	1003	0.14	84
MMC, 0.30	A	185	311	4	500	0.64
	B	172	323	5	500	0.67
	Total	357	634	9	1000	0.65	29 #

Data for 3+21 Hour Treatments +S-9, Micronucleus Experiment - Female Donors

Treatment	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity
(µg/mL)							Based on RI (%)
Vehicle	A	77	365	58	500	0.96
	B	89	373	44	506	0.91
	C	85	381	60	526	0.95
	D	93	337	70	500	0.95
	Total	344	1456	232	2032	0.94	-
10	A	124	378	84	586	0.93
	B	84	345	71	500	0.97
	Total	208	723	155	1086	0.95	0
20	A	85	349	66	500	0.96
	B	89	375	77	541	0.98
	Total	174	724	143	1041	0.97	0
30	A	89	352	59	500	0.94
	B	70	373	65	508	0.99
	Total	159	725	124	1008	0.97	0
40	A	63	370	67	500	1.01
	B	97	461	75	633	0.97
	Total	160	831	142	1133	0.98	0
50	A	84	384	42	510	0.92
	B	72	366	62	500	0.98
	Total	156	750	104	1010	0.95	0 #
60	A	118	370	46	534	0.87
	B	89	363	48	500	0.92
	Total	207	733	94	1034	0.89	6
70	A	120	386	48	554	0.87
	B	116	347	37	500	0.84
	Total	236	733	85	1054	0.86	9
80	A	134	340	26	500	0.78
	B	141	399	33	573	0.81
	Total	275	739	59	1073	0.8	15 #
90	A	145	353	35	533	0.79
	B	159	319	22	500	0.73
	Total	304	672	57	1033	0.76	19
100	A	250	325	20	595	0.61
	B	249	389	48	686	0.71
	Total	499	714	68	1281	0.66	30 #
CPA, 3.00	A	205	291	4	500	0.6
	B	202	290	8	500	0.61
	Total	407	581	12	1000	0.61	36
CPA, 5.00	A	326	272	3	601	0.46
	B	294	203	3	500	0.42
	Total	620	475	6	1101	0.44	53 #
CPA, 7.00	A	291	231	0	522	0.44
	B	310	190	1	501	0.38
	Total	601	421	1	1023	0.41	56

Data for 24+24 Hour Treatments -S-9, Micronucleus Experiment - Female Donors

Treatment	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity
(µg/mL)							Based on RI (%)
Vehicle	A	65	298	166	529	1.19
	B	64	350	151	565	1.15
	C	48	333	129	510	1.16
	D	50	333	117	500	1.13
	Total	227	1314	563	2104	1.16	-
10	A	67	341	92	500	1.05
	B	51	390	160	601	1.18
	Total	118	731	252	1101	1.12	3
20	A	56	327	118	501	1.12
	B	48	354	98	500	1.1
	Total	104	681	216	1001	1.11	4
25	A	62	384	100	546	1.07
	B	64	354	92	510	1.05
	Total	126	738	192	1056	1.06	8
30	A	57	339	106	502	1.1
	B	54	393	94	541	1.07
	Total	111	732	200	1043	1.09	6 #
35	A	54	397	79	530	1.05
	B	56	375	69	500	1.03
	Total	110	772	148	1030	1.04	11
40	A	53	366	81	500	1.06
	B	69	383	58	510	0.98
	Total	122	749	139	1010	1.02	12
42.5	A	71	352	77	500	1.01
	B	70	433	125	628	1.09
	Total	141	785	202	1128	1.05	9
45	A	97	429	104	630	1.01
	B	84	347	81	512	0.99
	Total	181	776	185	1142	1	13
47.5	A	120	393	97	610	0.96
	B	99	327	74	500	0.95
	Total	219	720	171	1110	0.96	17
50	A	120	319	61	500	0.88
	B	112	330	58	500	0.89
	Total	232	649	119	1000	0.89	24 #
55	A	181	265	64	510	0.77
	B	240	231	30	501	0.58
	Total	421	496	94	1011	0.68	42 #
60	A	354	124	22	500	0.34
	B	312	168	20	500	0.42
	Total	666	292	42	1000	0.38	68
VIN, 0.04	A	271	159	73	503	0.61
	B	220	199	91	510	0.75
	Total	491	358	164	1013	0.68	42 #

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

# Highlighted concentrations selected for analysis

Conclusions:

It is concluded that ZMBT; Zinc di(benzothiazol-2-yl) disulphide did induce micronuclei in cultured human peripheral blood lymphocytes following 3+21 hour treatment in the absence and presence of an Aroclor induced rat liver metabolic activation system (S-9) and 24+24 hour treatment in the absence of S-9. The maximum concentrations analysed were limited by solubility in the primary vehicle (3+21 hour treatment in the presence of S-9) or by toxicity (3+21 hour and 24+24 hour treatments in the absence of S-9).

Executive summary:

In vitro micronucleus assay test (Covance Laboratories Ltd., Study 8417384, 2020) was performed to evaluate the clastogenic and aneugenic potential of ZMBT by examining its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes. The test was conducted according to OECD test guideline 487 and in compliance with GLP.

Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9 mix). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide. The highest concentrations tested in the Micronucleus Experiment were either limited by solubility in the primary vehicle (3+21 hour treatment in the presence of S-9) or by toxicity (3+21 hour and 24+24 hour treatments in the absence of S-9). Concentrations to be tested were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatment of cells for 3+21 hours in the absence of S-9 mix resulted in frequencies of micronucleated binucleate (MNBN) cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (47.5 and 55 µg/mL, giving 18% and 47% reduction in Replication Index (RI), respectively). The increases observed exceeded the normal range in a single culture at 47.5 µg/mL and in both replicate cultures at 55 µg/mL.

Treatment of cells for 3+21 hours in the presence of S-9 mix resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (80 and 100 µg/mL, giving 15% and 30% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations.

Treatment of cells for 24+24 hours in the absence of S-9 mix resulted in frequencies of MNBN cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (50 and 55 µg/mL, giving 24% and 42% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations.

A statistically significant linear trend was observed in all treatments, indicating a positive results.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of MNBN cells in these cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid.

It is concluded that ZMBT did induce micronuclei in cultured human peripheral blood lymphocytes. The maximum concentrations analysed were limited by solubility in the primary vehicle (3+21 hour treatment in the presence of S-9 mix) or by toxicity (3+21 hour and 24+24 hour treatments in the absence of S-9 mix).

Genetic toxicity in vivo

Description of key information

The genotoxic potential of ZMBT was evaluated in an in vivo bone marrow chromosome aberration assay (Mohanan, 2000) with limited documentation, not in line with current guidelines. Swiss albino mice (4 animals per group) were treated once with ca. 24, 43 and 96 mg/kg bw test substance, concurrent solvent control (cotton seed oil) and positive control (methyl methane sulphonate, 200 mg/kg) via intraperitoneal injection. Colchicine (20 µg/kg) was administered 90 minutes before scheduled sacrifice. All animals were sacrificed 36 hours after test sample injection. Bone marrow cells from both femora were prepared, fixed and stained. The incidences of chromatid and chromosome gaps and breaks documented in all treatment groups were comparable to the solvent control. The authors concluded that ZMBT did not induce structural chromosomal aberrations in bone marrow cells of Swiss mice under the experimental conditions used.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

limited but acceptable documented publication which meets basic scientific principles; however study design not in line with current guidelines (e.g. sampling time 1.5 normal cell cylce lengh, preparation time after colcemid or colchicine application ca. 3 to 5 h for mice, 5 animals per sex and dose group, measure of cytotoxicity)

Principles of method if other than guideline:

other: chromosomal aberration assay in Swiss albino mice

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

provider: National Organic Chemical Industries Ltd., Chennai

Species:

mouse

Strain:

Swiss

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: weaning Swiss albino mice
- Weight at study initiation: 16 to 20 g
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2°C

Route of administration:

intraperitoneal

Vehicle:

cotton seed oil, 1 mL/kg bw

Details on exposure:

90 minutes before sacrifice the animals were treated with colchicine (20 µg/kg bw) to arrest mitosis

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

36 h

Dose / conc.:

24 mg/kg bw (total dose)

Remarks:

according to 0.480 mg/20 g animal

Dose / conc.:

43 mg/kg bw (total dose)

Remarks:

according to 0.86 mg/20 g animal

Dose / conc.:

96 mg/kg bw (total dose)

Remarks:

according to 1.920 mg/20 g animal

No. of animals per sex per dose:

4 per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

methyl methane sulfonate (200 mg/kg bw)

Tissues and cell types examined:

bone marrow from femur

Details of tissue and slide preparation:

Both femora were removed through the pelvic bone, just below the knee. The bones were freed from muscles, and the proximal ends of the femora were carefully shortened with scissors. Saline (0.5 ml) was aspirated into the disposable syringe, and subsequently, the needle was inserted a few millimeters into the bone marrow canal. The bone marrow was flushed into centrifuge tubes, mixed thoroughly and were centrifuged. The cell button was collected, mixed with hypotonic solution (0.075 M KCl), and incubated for 20 min at 37°C. The samples were centrifuged again, and were than fixed with fixative (3:1 methanol acetic acid). Sample slides were prepared by flame drying and stained with Giemsa stain. Microscopic analysis was performed by bright field in oil under a light microscope. Evidence of chromosomal abnormalities was then evaluated; 100 well-spread metaphases were analyzed per animal per dose group, solvent control and positive control.

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Chromosome number: number of chromosomes did not vary (no additional data)

The incidences of chromosome/chromatide abnormalities was comparable between treamtent groups and solvent control.

The postivie control induced significant number of chromosomal aberrations.

The authors conclude that the administration of ZMBT at different dose levels in Swiss mice did not increase the frequency of structural chromosomal aberrations and thus ZMBT did not induce structural chromosome aberrations in the bone marrow cells of Swiss mice under the experimental conditions used.

Table: Incidence of chromosomal aberrations after single ZMBT administration in the bone marrow of Swiss mice

		Average Abnormalities/100 plates
		Chromatid*		Chromosome*
Group	Dose/animal (µg)	Gaps	Breaks	Gaps	Breaks	Other changes*
I	1920	2	0	1	0	5
II	960	1	1	0	0	3
III	480	1	0	0	0	5
IV	solvent control 1.0 ml	2	1	1	0	7
V	positive control 4.0 mg**	17	6	11	8	19

* no additional data recorded

** methyl methane sulfonate (200 mg/kg bw)

Executive summary:

The genotoxic potential of ZMBT was evaluated in an in vivo bone marrow chromosome aberration assay with limited documentation not in line with current guidelines. Swiss albino mice (4 animals per group) were administered with ca. 24, 43 and 96 mg/kg bw test substance, concurrent solvent control (cotton seed oil) and positive control (methyl methane sulphonate, 200 mg/kg) once via intraperitoneal injection. Clinical signs or other observations of toxicity were not recorded in the publication. Colchicine (20 µg/kg) was administered 90 minutes before scheduled sacrifice. All animals were sacrificed 36 hours after test sample injection. Bone marrow cells from both femora were prepared, fixed and stained. 100 well-spread metaphases were microscopically analysed for chromosomal aberrations (chromatic and chromosome gaps and breaks and other changes were recorded). No cytotoxicity parameters (e.g. mitotic index) were recorded. The incidences of chromatid and chromosome gaps and breaks documented in all treatment groups were comparable to the solvent control. The positive control methyl methane sulfonate (200 mg/kg bw) led to a distinct increase of chromatid and chromosome breaks. ZMBT did not induce structural chromosomal aberrations in bone marrow cells of Swiss mice under the experimental conditions used.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study with acceptable restrictions, (e.g. proportion of immature erythrocyte among total erythrocytes not evaluated, dose level: only a dose of 300 mg/ml was evaluated (no limit test))

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

4 instead 5 animals/sex used

Principles of method if other than guideline:

Method: other: in vivo bone marrow micronucleus test with deviations

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: 10 weeks

Route of administration:

intraperitoneal

Vehicle:

corn oil; a good suspension was obtained at all levels tested; the test article was prepared fresh daily;

Details on exposure:

intraperitoneal injection volume of test article, positive control and negative control (corn oil) at a constant volume of 20 mL/kg bw.

Duration of treatment / exposure:

single dose of 300 mg/kg bw and
two doses of 300 mg/kg bw (0 and 24 hours)
The time of sacrifice and cell harvest were determined from initial treatment.

Frequency of treatment:

once or twice

Post exposure period:

30 and 48 hours in Single Dose group 1 and 2
48 and 72 hours in experiment 2 in Multiple Dose group 1 and 2
see Table 1

Dose / conc.:

300 mg/kg bw/day

Remarks:

single intraperitoneal injection

Dose / conc.:

600 mg/kg bw/day

Remarks:

splitted in two intraperitoneal doses of 300 mg/kg bw each, separated by 24 hours

No. of animals per sex per dose:

8 per dose (4 males and 4 females)

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control article triethylenemelamine, was administered intraperitoneally in 0.9% saline to a separate group of mice (4 males and 4 females) at a dose of 0.5 mg/kg bw.

Tissues and cell types examined:

polychromatic erythrocytes prepared from the femur of treated mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Results of a dose range finding experiment:
Dose Range Finding Study:
In a preliminary dose range finding study mercaptobenzothiazole was administered intraperitoneally once daily for two consecutive days to groups of four mice (two males, two females each) at 16.6, 50, 166.6, 500 and 1,666.6 mg/kg of body weight. Mortality and clinical signs were observed up to 48 hours following the second dose at which time remaining animals were sacrificed. Based on mortality and severity of signs a dose level of 300 mg/kg bw was selected for the micronucleus assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:
Two slides were prepared from the bone marrow of the femurs, stained and coded for each animal in the assay.
The femur was opened carefully at the proximal end with a scissors until a small opening to the marrow canal became visible. A 1 ml tuberculin syringe filled vith approximately 0.2 ml fetal calf serum was inserted into the bone and the bone marrow was gently flushed (to assure maximum dispersion) into
1.0 ml of fetal calf serum in a 3 ml conical centrifuge tube. The femur was flushed with fetal calf serum until all the marrow was out and the bone appeared almost transparent. If necessary, the distal end was opened and flushed. The suspension was centrifuged at 1000 rpm for five minutes. Following preparation of the smears, they vere dipped in absolute methanol and allowed to air dry overnight.
STAINING:
Fixed in absolute methanol - 5 minutes and air dried
Stain 20 minutes in a 5% Giemsa stain

METHOD OF ANALYSIS:
Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE per animal.

OTHER:
All animals were sacrificed by cervical dislocation and their femurs removed.
Slides WEre coded randomly by study nUlllber and letter designation. The code
was kept on a separate sheet in the .ponsor's file until the slides were
evaluated. Following ~valuation, the slides were decoded and the code sheet
was placed in the notebook. These procedures are carried out by • technician
not involved in the actual scoring of the .tudy.

Evaluation criteria:

Assessment of a test article as positive is based upon its ability to produce a statistically significant increase in the number of micronuclei in polychromatic erythrocytes as compared, to the negative control. A t-test was used to evaluate pairwise treatment groups with the negative control for statistically significant increases in the number of micronuclei.

Statistics:

A t-test was used to evaluate pairwise treatment groups with the negative control for statistically significant increases in the number of micronuclei per 1000 polychromatic erythrocytes (PCE)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Pre-experiment (toxicity)

Immediate mortality was observed in four of four animals at the 1,666.6 mg/kg dose level. At 500 mg/kg, two females died within four hours of the first dose. The remaining two males had decreased muscle tone and activity and ptosis. Total death occurring at this level was two of four animals. At 166.6 and 50 mg/kg, the majority of the animals showed no signs, but an occasional animal had decreased tone. No signs were observed at the 16.6 mg/kg level. No mortality occurred at the 166.6, 50 and 16.6 mg/kg levels.

Based on the results of the dose range finding study, the dose selected for the Micronucleus assay was 300 mg/kg bw.

Main experiment:

Pharmacological Effects of Treatment

The following signs were observed at the first dose: prostration, hypoactivity, hypernea, ptosis, tremors upon stimulation and an occasional animal exhibited a loss of righting. Observations at 4 and 24 hours following the first dose included ptosis with no other visible signs in all treated animals. No mortality occurred in the study.

Genotoxicity (see Table 2):

The results for test article mercaptobenzothiazole were negative in the micronucleus test at a dose level of 300 mg/kg in the single dose groups and with a second dose of 300 mg/kg in the multiple dose group administered in a split dose regimen. The test material did not produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all animals administered mercaptobenzothiazole were within the normal historical range of spontaneous micronuclei incidence.

Table 2: Micronuclei/1000 PCE

	negative control	positive control	Single Dose, 300 mg/kg bw		Multiple Dose, 2 x 300 mg/kg bw
animal number	corn oil	TEM	S1	S2	M1	M2
male	20 mg/kg bw	0.5 mg/kg bw	30 hrs	48 hrs	48 hrs	72 hrs
1	0	20	1	2	0	0
2	1	43	1	1	0	0
3	0	68	0	0	0	0
4	0	28	1	1	0	0
female
5	0	20	0	1	0	0
6	0	40	0	0	0	1
7	0	28	0	0	0	0
8	0	17	0	0	0	0
mean	0.125	34.125	0.375	0.625	0.00	0.125
't' value	-	5.906**	1.128	1.716	1.000	0

** Denotes statistical significance at the 0.01 level

Conclusions:

Interpretation of results: negative

Executive summary:

The genotoxic potential of MBT was evaluated in an in vivo micronucleus assay with CD-1 mice (4 males and 4 females per group) and via intraperitoneal injection. Single dose group animals received 300 mg/kg bw of MBT and multiple dose group animals received MBT in a split dose regimen with two doses of 300 mg/kg bw each, separated by 24 hours. The positive control article triethylenemelamine, was administered intraperitonealy to a separate group of mice (4 males and 4 females) at a dose of 0.5 mg/kg. Thirty hours after treatment the positive control animals were sacrificed. The negative control animals were given two doses of corn oil separated by 24 hours and sacrificed 48 hours after the first dose. Single Dose Group I and Single Dose Group II were sacrificed at 30 and 48 hours, respectively after a single injection. Multiple Dose Group I and Multiple Dose Group II were given two doses of the test article separated by 24 hours and sacrificed at 48 and 72 hours, respectively after the initial injection.

Systemic availability of the test substance was indicated by occurrence of clinical signs after application. The following signs were observed at the first dose: prostration, hypoactivity, hypernea, ptosis, tremors upon stimulation and an occasional animal exhibited a loss of righting. Observations at 4 and 24 hours following the first dose included ptosis with no other visible signs in all treated animals. No mortality occurred in the study.

The positive control led to a distinct and statistically significant increase in micronuclei. The results for test article MBT were negative in the micronucleus test after single intraperitoneal treatment with 300 mg/kg bw (sacrifice 30 and 48 hour after dosing) and after two intraperitoneal treatments with 300 mg/kg bw, separated by 24 hours (sacrifice 48 and 72 hours after the first dosing). The test material did not produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all animals administered MBT were within the normal historical range of spontaneous micronuclei incidence.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: comparable to guideline study with acceptable restrictions, (e.g. proportion of immature erythrocyte among total erythrocytes not evaluated, dose level: only a dose of 300 mg/ml was evaluated (no limit test))

Justification for type of information:

Read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Pre-experiment (toxicity)

Immediate mortality was observed in four of four animals at the 1,666.6 mg/kg dose level. At 500 mg/kg, two females died within four hours of the first dose. The remaining two males had decreased muscle tone and activity and ptosis. Total death occurring at this level was two of four animals. At 166.6 and 50 mg/kg, the majority of the animals showed no signs, but an occasional animal had decreased tone. No signs were observed at the 16.6 mg/kg level. No mortality occurred at the 166.6, 50 and 16.6 mg/kg levels.

Based on the results of the dose range finding study, the dose selected for the Micronucleus assay was 300 mg/kg bw.

Main experiment:

Pharmacological Effects of Treatment

The following signs were observed at the first dose: prostration, hypoactivity, hypernea, ptosis, tremors upon stimulation and an occasional animal exhibited a loss of righting. Observations at 4 and 24 hours following the first dose included ptosis with no other visible signs in all treated animals. No mortality occurred in the study.

Genotoxicity (see Table 2):

The results for test article mercaptobenzothiazole were negative in the micronucleus test at a dose level of 300 mg/kg in the single dose groups and with a second dose of 300 mg/kg in the multiple dose group administered in a split dose regimen. The test material did not produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all animals administered mercaptobenzothiazole were within the normal historical range of spontaneous micronuclei incidence.

Table 2: Micronuclei/1000 PCE

	negative control	positive control	Single Dose, 300 mg/kg bw		Multiple Dose, 2 x 300 mg/kg bw
animal number	corn oil	TEM	S1	S2	M1	M2
male	20 mg/kg bw	0.5 mg/kg bw	30 hrs	48 hrs	48 hrs	72 hrs
1	0	20	1	2	0	0
2	1	43	1	1	0	0
3	0	68	0	0	0	0
4	0	28	1	1	0	0
female
5	0	20	0	1	0	0
6	0	40	0	0	0	1
7	0	28	0	0	0	0
8	0	17	0	0	0	0
mean	0.125	34.125	0.375	0.625	0.00	0.125
't' value	-	5.906**	1.128	1.716	1.000	0

** Denotes statistical significance at the 0.01 level

Conclusions:

This in vivo micronucleus assay with MBT with negative outcome supports the negative in vivo Chromosomal Aberration assay performed with ZMBT.

Executive summary:

The genotoxic potential of MBT was evaluated in an in vivo micronucleus assay with CD-1 mice (4 males and 4 females per group) and via intraperitoneal injection. Single dose group animals received 300 mg/kg bw of MBT and multiple dose group animals received MBT in a split dose regimen with two doses of 300 mg/kg bw each, separated by 24 hours. The positive control article triethylenemelamine, was administered intraperitonealy to a separate group of mice (4 males and 4 females) at a dose of 0.5 mg/kg. Thirty hours after treatment the positive control animals were sacrificed. The negative control animals were given two doses of corn oil separated by 24 hours and sacrificed 48 hours after the first dose. Single Dose Group I and Single Dose Group II were sacrificed at 30 and 48 hours, respectively after a single injection. Multiple Dose Group I and Multiple Dose Group II were given two doses of the test article separated by 24 hours and sacrificed at 48 and 72 hours, respectively after the initial injection.

Systemic availability of the test substance was indicated by occurrence of clinical signs after application. The following signs were observed at the first dose: prostration, hypoactivity, hypernea, ptosis, tremors upon stimulation and an occasional animal exhibited a loss of righting. Observations at 4 and 24 hours following the first dose included ptosis with no other visible signs in all treated animals. No mortality occurred in the study.

The positive control led to a distinct and statistically significant increase in micronuclei. The results for test article MBT were negative in the micronucleus test after single intraperitoneal treatment with 300 mg/kg bw (sacrifice 30 and 48 hour after dosing) and after two intraperitoneal treatments with 300 mg/kg bw, separated by 24 hours (sacrifice 48 and 72 hours after the first dosing). The test material did not produce a statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all animals administered MBT were within the normal historical range of spontaneous micronuclei incidence.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

ZMBT did not induce structural chromosomal aberrations in bone marrow cells of Swiss mice under the experimental conditions used.

Additional information

In vitro data for ZMBT

A Bacterial reverse mutation test was conducted to examine the potential of ZMBT to cause gene mutation. The study was conducted in accordance to OECD test guideline 471 and in compliance with GLP. Five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation (S9) were exposed ZMBT in two separate experiments in 0.5% methyl cellulose as vehicle. The treatments were performed up to the maximum recommended concentration (5000 µg/plate) according to current regulatory guidelines. In Mutation Experiment 1, evidence of toxicity in the form of a reduction in the number of revertants was observed at 5000 μg/plate in strain TA102 in the absence and presence of S-9. In Mutation Experiment 2, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 5000 μg/plate in strains TA98 and TA102 in the presence of S-9 and at 2500 μg/plate and above in strain TA102 in the absence of S-9. The mean numbers of revertant colonies were comparable with acceptable ranges for vehicle control treatments, and were elevated by positive control treatments. It was concluded that ZMBT did not induce mutation in the Ames test when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate in the absence and in the presence of a rat liver metabolic activation system (S9).

The mutagenic potential of ZMBT was evaluated in a bacterial mutagenicity test (Monsanto Co. 1977). Although the study is reliable the test design of the study does not comply with the current guideline with regard to the kind of tester strains used. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. Treatment by the plate incorporation method was done in presence or absence of metabolic activation (S9-mix). A concentration range of 0.1 mg/plate to 500 µg/plate was evaluated. Toxicity was noted in tester strain TA 1538 at 500µg/plate without metabolic activation. No mutagenic response was noted in any of the tester strains used in presence or absence of metabolic activation (S9-mix). The authors concluded that the test substance did not induce a mutagenic response in any of the tester strains evaluated under the experimental conditions used.

An in vitro mammalian cell gene mutation test was performed to access the ability of ZMBT to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in mouse lymphoma cells. The test was conducted according to OECD test guideline 476. Based on range-finder results showing toxicity the Mutation Experiment was conducted with ten concentrations of up to 50.00 μg/mL in the absence of S9 and up to 100.0 μg/mL in the presence of S9. Test solutions were formulated in anhydrous analytical grade DMSO. Following 3 hour treatment no statistically significant increases in mutant frequency (MF), compared to the vehicle control, were observed at any concentration analysed and there were no statistically significant linear trends, indicating a negative result. Seven days after treatment, the highest concentrations analysed were 35 μg/mL in the absence of S9 and 60 μg/mL in the presence of S-9, which gave 18% and 17% relative survival (RS), respectively. Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S9 and showed the expected results. It is concluded that ZMBT did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations, in the absence and presence of a rat liver metabolic activation system (S9).

An in vitro micronucleus assay test was performed to evaluate the clastogenic and aneugenic potential of ZMBT by examining its effects on the frequency of micronuclei in cultured human peripheral blood lymphocytes. The test was conducted according to OECD test guideline 487 and in compliance with GLP. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9 mix). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide. The highest concentrations tested in the Micronucleus Experiment were either limited by solubility in the primary vehicle (3+21 hour treatment in the presence of S-9) or by toxicity (3+21 hour and 24+24 hour treatments in the absence of S-9). Concentrations to be tested were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatment of cells for 3+21 hours in the absence of S-9 mix resulted in frequencies of micronucleated binucleate (MNBN) cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (47.5 and 55 µg/mL, giving 18% and 47% reduction in Replication Index (RI), respectively). The increases observed exceeded the normal range in a single culture at 47.5 µg/mL and in both replicate cultures at 55 µg/mL. Treatment of cells for 3+21 hours in the presence of S-9 mix resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (80 and 100 µg/mL, giving 15% and 30% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations. Treatment of cells for 24+24 hours in the absence of S-9 mix resulted in frequencies of MNBN cells which were significantly (p≤0.01) higher than those observed in concurrent vehicle controls at the highest two concentrations analysed (50 and 55 µg/mL, giving 24% and 42% reduction in RI, respectively). The increases observed exceeded the normal range in both replicate cultures for both concentrations. A statistically significant linear trend was observed in all treatments, indicating a positive results. All acceptance criteria were considered met and the study was therefore accepted as valid.

It is concluded that ZMBT did induce micronuclei in cultured human peripheral blood lymphocytes.

In vitro data for MBT

As ZMBT, MBT was shown to be negative in gene mutation test in bacteria and mammalian cells. In a chromosome aberration assay with CHL cells an induction of polyploidy cells and  endoreduplications was noted. No endoreduplications were noted in the solvent control (Matsuoka 2005). Furthermore, in a limited chromosome aberration assay with CHO cells an increase in aberrant cells was observed (NTP 1988) and in a limited sister chromatid exchange (SCE) assay a relative increase in SCE's was noted in presence of metabolic activation (NTP 1988). However, the relevance of these finding is questionable because of presumed high toxicity indicated by a significant chemically induced cell cycle delay and the lacking of a dose-response relationship.

In vivo data for ZMBT

The genotoxic potential of ZMBT was evaluated in an in vivo bone marrow chromosome aberration assay with limited documentation (Mohanan, 2000). Swiss albino mice (4 animals per group) were administered with ca. 24, 43 and 96 mg/kg bw test substance, concurrent solvent control (cotton seed oil) and positive control (methyl methane sulphonate, 200 mg/kg) once via intraperitoneal injection. Clinical signs or other observations of toxicity were not recorded in the publication. Colchicine (20 µg/kg) was administered 90 minutes before scheduled sacrifice. All animals were sacrificed 36 hours after test sample injection. Bone marrow cells from both femora were prepared, fixed and stained. 100 well-spread metaphases were microscopically analysed for chromosomal aberrations (chromatic and chromosome gaps and breaks and other changes were recorded). No cytotoxicity parameters (e.g. mitotic index) were recorded. The incidences of chromatid and chromosome gaps and breaks documented in all treatment groups were comparable to the solvent control. The positive control methyl methane sulfonate (200 mg/kg bw) led to a distinct increase of chromatid and chromosome breaks. ZMBT did not induce structural chromosomal aberrations in bone marrow cells of Swiss mice under the experimental conditions used.

Supporting in vivo information of MBT

No genotoxic effects of MBT were noted in an in vivo micronucleus assay with intraperitoneal injection of MBT to CD-1 mice (4 males and 4 females per group). Single dose group animals received 300 mg/kg bw of MBT (sacrifice 30 and 48 hours after dosing) and multiple dose group animals received MBT in a split dose regimen with two doses of 300 mg/kg bw each, separated by 24 hours (sacrifice 48 and 72 hours after initial injection). Systemic availability of the test substance was indicated by occurrence of clinical signs after application. No mortality occurred in the study. The test material MBT did not produce statistically significant increases in the number of micronuclei per 1000 polychromatic erythrocytes in the treated versus the control group. In addition to these criteria, all micronucleus frequencies in animals treated with MBT were within the normal historical range of spontaneous micronuclei incidence (CMA 1984). In addition to the micronucleus assay, a dominant lethal test with Sprague-Dawley rats indicated no dominant lethal effects of the test substance MBT (CMA 1989).

Information on zinc

Zn2+ is an essential trace element for human nutrition and ubiquitous in biological systems including humans. The human body has efficient mechanisms, both on systemic and cellular levels, to maintain zinc homeostasis over a broad exposure range. Consequently, zinc has a rather low toxicity. On the other hand, zinc deficiency is a condition with broad occurrence and potentially profound impact (Plum et al., 2010). Toxicity and impacts on human health of zinc has been extensively evaluated by the EU (EU RAR, 2004), the Agency for Toxic Substances and Disease Registry (ATSDR, 2006) and in reviews of Plum et al. (2010), and Chasapis et al. (2020). Zinc is thus included in the assessment of ZMBT based on these assessments.

As discussed in the EU risk assessment (2004) there are several in vitro and in vivo genotoxicity data available for several zinc compounds. The available data indicate that genotoxicity results vary widely (EU risk assessment 2004, MAK 2009). However, there is no clear evidence from the available data that zinc is genotoxic in vivo (EU risk assessment 2004).

In conclusion and based on the available information on genetic toxicity for ZMBT and for the hydrolysis product MBT it can be concluded that ZMBT does not exert a mutagenic potential in vivo. MBT and ZMBT both show no potential to induce gene mutations in bacteria or mammalian cells in vitro. Furthermore, both substances were shown to exert a clastogenic potential in vitro, that was not verified in vivo, shown by negative outcome of in vivo micronucleus/chromosomal aberration assays with intraperitoneal injection of the substances. Thus, MBT and ZMBT can be considered as not genotoxic in vivo.

Justification for classification or non-classification

No classification is required according to the classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).