Zinc bis(dibenzyldithiocarbamate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Sep 2018 to 20 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dibenzyldithiocarbamate)

EC Number:

238-778-0

EC Name:

Zinc bis(dibenzyldithiocarbamate)

Cas Number:

14726-36-4

Molecular formula:

C30H28N2S4Zn

IUPAC Name:

zinc bis(dibenzyldithiocarbamate)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar outbred (Crl:WI(Han)) (SPF)

Details on species / strain selection:

For this study, rats were chosen as test system, because this animal species is normally used in toxicity studies of this type.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 201.9 – 266.1 gram
- Assigned to test groups randomly: yes
- Housing: The animals were housed two to five animals per cage. All animals were housed in Makrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly. Due to a staggered start, not all animals were dosed at once. To avoid exposure of the animals not yet dosed, the dosed animals were placed in a different cage. As a consequence, the first animals per group that were dosed, were housed individually until the second animals of the same group had been dosed (time between dosing of animals of the same group was ca. 2-3 hours). Also, the last animals per group that were necropsied, were housed individually for a short period of time (ca. 2-3 hours). Animals treated with the positive controls MMS and 2-AAF were housed in filter top cages (two to three animals per cage) after dosing.
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England).
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study. Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC) was supplied by N.V. Vitens.
- Acclimation period: 12-14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 – 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 September 2018 To: 20 September 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 8, 40 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw/day
- Lot/batch no. (if required): A1701528
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For each day of the study and for each test substance group, the appropriate amount of test substance was weighed in a glass bottle. Each dosing day, the corresponding amount of corn oil was added to obtain the final concentration of the test substance in corn oil. Before dosing, the suspension was stirred for at least 30 minutes, until visual homogeneity was obtained. All suspensions were continuously stirred on a magnetic stirrer during the dosing procedure, in order to maintain the homogeneity of the test substance in the vehicle.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

Twice, with an interval between the first and second dose of ca. 20 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose. Surplus animals were included in group 1 and 2 to replace the duodenum collected from animals 2 (group 1) and 12 (group 2).

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene (liver) and methylmethanesulfonate (glandular stomach & duodenum)
- Route of administration: gavage (2-acetylaminofluorene and methylmethanesulfonate)
- Doses / concentrations: dosed once with methylmethanesulfonate: 40 mg/kg bw (nominal); 2-acetylaminofluorene: 50 mg/kg bw (nominal)

Examinations

Tissues and cell types examined:

glandular stomach, duodenum and liver

Details of tissue and slide preparation:

ISOLATION OF GLANDULAR STOMACH CELLS
All steps were performed on ice as much as possible. The forestomach was removed and preserved in formaldehyde and the glandular stomach was cut open and washed free from food and debris using ice-cold mincing buffer. The glandular stomach was immersed in fresh ice-cold mincing buffer again and incubated for 15-30 minutes. After incubation, the surface epithelia was gently scraped two times using a Teflon scrapper. This layer was discarded and the gastric mucosa was rinsed with the ice-cold mincing buffer. The stomach epithelia was carefully scraped 4-5 times (or more, if necessary) with a scrapper to release the cells. The cell suspension was strained with a 40 μm cell strainer to remove clumps and the remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

ISOLATION OF DUODENUM CELLS
All steps were performed on ice as much as possible. The duodenum was cut open and washed free from food and debris using ice-cold mincing buffer. Subsequently, the duodenum was minced with a pair of fine scissors to release the cells. The cell suspension was strained with a 500 μm netwell filter, followed by a 40 μm cell strainer to remove clumps. The remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

ISOLATION OF LIVER CELLS
All steps were performed on ice as much as possible. The collected portion of the left lateral liver lobe was washed in ice-cold mincing buffer until as much blood as possible had been removed. Subsequently, the portion was minced with a pair of fine scissors to release the cells. The cell suspension was strained with a 500 μm netwell filter, followed by a 40 μm cell strainer to remove clumps. The remaining suspension was centrifuged (3 min, 500 rpm, ca. 4°C). The supernatant was discarded except for a small volume to re-suspend the cells, followed by preparation of comet slides. The cell density and viability of the cells was determined by trypan blue exclusion.

PREPARATION OF COMET SLIDES
Microscopic slides were prepared by mixing an aliquot of the cell suspension with a low-melting agarose solution (0.5 % (w/v) in Phosphate Buffered Saline). Subsequently, this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Three slides per animal were prepared (one slide was kept in reserve). The slides were stored on a cold plate until the agarose had solidified. Subsequently, the coverslip was removed and the slide was incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 10% DMSO (v/v) and 1 % Triton X-100 (v/v), pH 10) overnight at 2-10 ºC. After incubation in lysis buffer, the slides were shortly rinsed in ice-cold electrophoreses buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13). Subsequently, slides were incubated in ice-cold electrophoresis buffer for 20 ± 1 min (unwinding), followed by electrophoresis (28 V and ca. 300 mA) for 30 ± 1 min in ice-cold electrophoresis buffer, while cooled on ice. The temperature of the electrophoresis buffer was measured at the start of unwinding, the start of electrophoresis and the end of electrophoresis. After incubation in neutralization buffer (0.4 M Tris in Milli-Q water, pH 7.5) for at least 5 min, slides were dehydrated by incubating in ethanol at room temperature and air-dried.

SLIDE ANALYSIS AND COUNTING
Slides were coded by a qualified person not involved in analyzing the slides to enable ‘blind’ scoring. Slides were stained with SYBR Gold, which was 10.000 times diluted with TE buffer (10 mM Tris and 1 mM Na2EDTA, pH ca. 7.0-7.5) and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software was used for the analysis of the slides. Seventy-five cells (randomly selected starting from the center of the slide) per slide and two slides per animal were analyzed to yield a total number of 150 cells per animal. Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was recorded.

Evaluation criteria:

ACCEPTANCE CRITERIA
The study was considered valid for the tissue if:
- the mean tail intensity of the negative control group was ≤ 20% (glandular stomach), ≤ 10% (duodenum) and ≤ 6% (liver).
- the group mean tail intensity of the group 5 (for MMS) or group 6 (for 2-AAF) demonstrated a statistically significant increase compared to the group mean of the negative control group (group 1)
- at least 150 cells from at least 2 slides were analyzed for all animals included in the group

EVALUATION AND INTERPRETATION OF THE RESULTS
A test substance is considered to be positive if:
- at least one dose level demonstrated a statistically significant increase compared to the negative control (group 1)
- the increase was dose-related when evaluated with a test for a linear trend

When both criteria were met, the test substance was considered to be able to induce DNA strand breakage in the tissue evaluated, under the conditions used in this study.

A test substance was considered to be negative if:
- none of the dose levels demonstrated a statistically significant increase compared to the negative control (group 1)
- there was no dose-related increase when evaluated with a test for a linear trend
- direct or indirect evidence of exposure of, or toxicity to, the target tissue was demonstrated
When all of these criteria were met, the test substance was considered not to be able to induce DNA strand breakage in the tissue evaluated, under the conditions used in this study.

There is no requirement for verification of a clearly positive or negative response. In case the response was neither clearly negative nor clearly positive (i.e. not all the criteria listed above are met) the results will be evaluated by expert judgement and/or further investigations will be conducted, if scientifically justified (in consultation with the Sponsor).

Statistics:

see "Any other information on materials and methods incl. tables"

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: applicable for glandular stomach and liver

Test results 2

Sex:

male

Genotoxicity:

other: inconclusive

Toxicity:

no effects

Vehicle controls validity:

not valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Remarks on result:

other: applicable for duodenum

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Clinical signs and mortality
No mortality was observed and no treatment-related clinical signs were observed in the animals during the study period.

Body weights
Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. Possible effects on mean body weight following treatment with the test substance were not determined.

Comet assay
- Viability of isolated cells:
After necropsy, glandular stomach, duodenum and liver cells were isolated by scraping or mincing the cells. Subsequently, comet slides were prepared. The percentage viability of the isolated glandular stomach cells was 77%, 72%, 78% and 76% for the negative control (corn oil), low, mid and high concentration of the test substance respectively, whereas the percentage viability of the isolated liver cells was 80%, 76%, 85% and 77%, for the negative control, low, mid and high concentration of the test substance, respectively. As the percentage viability of the isolated duodenum cells in two animals was extremely low (26% and 12% for animal no. 2 and 12, respectively), the two surplus animals were included in the study to replace the duodenum of these animals. Data for duodenum from animals 2 and 12 were excluded from calculations of the group means and from further evaluation. The percentage viability of the isolated duodenum cells was 93%, 94%, 90% and 94% for the negative control, low, mid and high concentration of the test substance, respectively. Based on the observed viability, all cell suspensions were considered suitable for the comet assay.

- DNA damage:
For each animal, 75 cells per slide and two slides per animal were analysed (i.e. 150 cells per animal per tissue in total). For glandular stomach and liver, all acceptance criteria for a valid test were met. The positive control substances MMS (group 5) and 2-AAF (group 6) demonstrated a statistically significant increase in tail intensity compared to the negative control (group 1, corn oil), respectively (p-value: <0.0001 for both tissues) and the group mean tail intensity of the negative control (corn oil) was <20% and <6% for glandular stomach and liver, respectively. For duodenum, the acceptance criteria were not met. The group mean tail intensity of the negative control was >10% (28.69%). The higher tail intensity observed in the negative control and some of the treatment groups seemed to be associated with an increased number of hedgehog cells. This suggests that the higher tail intensity may be caused by cytotoxicity, which in turn may have been induced during processing of the cells. The positive control substance MMS (group 5) did not demonstrate a statistically significant increase in tail intensity compared to the negative control group (group 1, corn oil) for duodenum. This means that the data obtained for duodenum are not valid and therefore no conclusions can be drawn for this organ.

Tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity in the glandular stomach and liver at any of the concentrations tested.

In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available fraction of the test substance and its metabolites, whereas the glandular stomach and the duodenum was evaluated to detect the genotoxic potential of the test substance at the ‘site of first contact’. Since the data for duodenum are not valid, the ‘site of first contact’ is covered only by the glandular stomach.

Details on tail intensities measure are presented in 'Any other information on results incl. tables'.

Any other information on results incl. tables

Group	Test substance and concentration	Tail intensity glandular stomach cells	Tail intensity duodenum cells	Tail intensity liver cells
1	Negative control (corn oil)	12.57 ± 5.54	28.69 ± 19.24	0.21 ± 0.25
2	80 mg/kg-bw test substance (low)	9.24 ± 1.37	24.82 ± 19.41	0.21 ± 0.28
3	400 mg/kg-bw test substance (mid)	11.34 ± 1.75	10.98 ± 3.73	0.26 ± 0.33
4	2000 mg/kg-bw test substance (high)	12.47 ± 2.73	16.96 ± 16.37	0.09 ± 0.10
5	Positive control (stomach, duodenum, MMS)	34.15 ± 1.88a	25.31 ± 1.90b	N.D.
6	Positive control (liver, 2-AAF)	N.D.	N.D.	17.46 ± 3.72c

 

a Statistically significantly increased compared to concurrent negative control, Students t-test (unpaired): p<0.0001

b Not statistically significantly increased compared to concurrent negative control, Mann Whitney’s (nonparametric) p-value: 0.6905

c Statistically significantly increased compared to concurrent negative control, Students t-test (unpaired) with transformed data: p<0.0001

N.D. Not determined

 

Applicant's summary and conclusion

Conclusions:

Although the data obtained from the duodenum did not fulfill the validity criteria, data were obtained from glandular stomach covering the ‘site of first contact’ and liver as the metabolizing organ, for evaluation of the genotoxic potential of the test substance. Hence, it can be concluded that, under the conditions used in this study, the test substance did not show any indication of induction of primary DNA damage in glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day. The data for the duodenum are inconclusive. However, considering all available information in a separate expert statement (included as attachment to this RSS), it can be concluded that ZBEC is not genotoxic.

Executive summary:

In this GLP compliant in vivo comet assay performed according to OECD guideline 489, the test substance was examined for its potential to cause primary DNA damage (such as single and double strand DNA breaks, alkali labile sites and incomplete repair sites) in glandular stomach, duodenum and liver cells of rats, following oral (gavage) administration of the test substance.

Male rats (n=5) were orally administered (by gavage, dosing volume 10 mL/kg bw/day) three concentrations (80, 400 and 2000 mg/kg bw/day) of the test substance or vehicle (corn oil) on two successive days, with ca. 20 h interval between the first and second dose. The second dose was administered ca. 3 h before scheduled sacrifice. This exposure regimen meets the OECD guideline 489 requirements to sample 2-6 h and 16-26 h after administration of the test substance in the same animal. Maximum dose levels were based on an oral (gavage) dose range finding study (not part of this study) and read-across data on acute toxicity provided by the sponsor. Positive control animals for glandular stomach and duodenum were orally administered (by gavage) once with methyl methanesulfonate (MMS, 40 mg/kg bw) 2-6 h prior to sacrifice. Positive control animals for liver were orally administered (by gavage) once with 2-acetylaminofluorene (2-AAF, 50 mg/kg bw) 12-16 h prior to sacrifice, respectively.

All animals survived until scheduled sacrifice. No treatment-related clinical abnormalities were observed. There were no statistically significant effects on mean body weight following treatment.

Approximately 3 h after the last oral dose, the glandular stomach, duodenum and part of the left lateral liver lobe were collected, followed by mincing to obtain single cell suspensions and preparation of comet slides. Based on the observed viability, all cell suspensions were considered suitable for the comet assay, except for the duodenum in two animals (animal no. 2 and 12). Therefore, the two surplus animals were included in the study to replace the duodenum of animals no. 2 and 12.

Tail intensity (i.e. the percentage DNA in the ‘tail’ of the comet) was used as a measure for primary DNA damage in the comet assay. Seventy-five cells per slide and two slides per animal were analyzed (i.e. in total 150 cells per animal per tissue). The median of each slide was calculated and the mean of the two medians was calculated per animal. Finally, the group mean of the individual animal values was calculated.

For glandular stomach and liver, all acceptance criteria for a valid test were met. The positive control substances MMS (group 5) and 2-AAF (group 6) demonstrated a statistically significant increase in tail intensity compared to the negative control (group 1, corn oil), respectively (p-value: <0.0001 for both tissues) and the group mean tail intensity of the negative control (corn oil) was <20% and <6% for glandular stomach and liver, respectively. For duodenum, the acceptance criteria were not met. The group mean tail intensity of the negative control was >10% (28.69%). The higher tail intensity observed in the negative control and some of the treatment groups seemed to be associated with an increased number of hedgehog cells. This suggests that the higher tail intensity may be caused by cytotoxicity, which in turn may have been induced during processing of the cells. The positive control substance MMS (group 5) did not demonstrate a statistically significant increase in tail intensity compared to the negative control group (group 1, corn oil) for duodenum. This means that the data obtained for duodenum are not valid and therefore no conclusions can be drawn for this organ.

Tail intensity of the test substance was comparable to the negative control (corn oil) and did not demonstrate a statistically significant increase in tail intensity in the glandular stomach and liver at any of the concentrations tested.

In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available fraction of the test substance and its metabolites, whereas the glandular stomach and the duodenum was evaluated to detect the genotoxic potential of the test substance at the ‘site of first contact’. Since the data for duodenum are not valid, the ‘site of first contact’ is covered only by the glandular stomach.

It can be concluded that, under the conditions used in this study, the test substance did not show any indication of induction of primary DNA damage in glandular stomach and liver cells of male rats after oral administration up to 2000 mg/kg bw/day. The data for the duodenum are inconclusive.

In the attached expert statement all available information is considered and it is concluded that it is highly unlikely that ZBEC will be mutagenic, either via systemic toxicity or via site of contact. From the available information it can be concluded that due to the size of the substance, the potential to cross biological membranes is limited, limiting the potential to damage DNA. In addition, no mutagenicity was observed in the other two organs examined in the in vivo Comet assay, neither via site of contact or via systemic toxicity. The absence of (eco)toxicity in available (eco)toxicity studies supports this, as well as information from the structurally similar compound ZDBC. Therefore, the available information is sufficient to conclude that ZBEC is not genotoxic.