Polysulfides, di-tert-dodecyl

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:

August - November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

Deviations:

yes

Remarks:

For the duodenum, median of percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound of negative historical control data : it constitutes a deviation according to OECD test guideline.

Principles of method if other than guideline:

The purpose of the in vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988), is to identify those agents which induce DNA damage such as single or double DNA strand breaks 10(SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites in compliance with the OECD guideline 489 (2016). The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Polysulfides, di-tert-dodecyl

EC Number:

270-335-7

EC Name:

Polysulfides, di-tert-dodecyl

Cas Number:

68425-15-0

Molecular formula:

not applicable

IUPAC Name:

di (alkyl C11-C13 Branched, C12 Rich), polysulfure S3-S5 rich

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is the species recommended by OECD guideline for this test.

No difference in terms of toxicity was noted between male and female rats in the previous in vivo
genotoxicity study. At the Sponsor’s request, the study was performed in male rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: 190 g to 210 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in polypropylene cages
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 19330), ad libium
- Water (e.g. ad libitum): 30-70 %,
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %,
- Air changes (per hr): not precised, but ventilated system
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose volume : 5 mL/kg b.w.
Form administered : solution

In the main genotoxicity assay, a solution of the test item at the concentrations of 400 mg/mL was prepared, giving a final dose of 2000 mg/kg, when administered at 5 mL/kg. The lowest concentrations of 224 and 82 mg/mL were obtained by successive dilution in corn oil for final dose of 1120 and 410 mg/kg.

Duration of treatment / exposure:

2 daily treatments at 24-hour intervals

Frequency of treatment:

2 daily treatments at 24-hour intervals

Post exposure period:

2-6 hours after the second treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

410 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 120 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/group, except for the high dose group : 7 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

methylmethane sulfonate (Aldrich, batch MKBX5165V) in sterile water (Fresenius, Batch 13PEP301), 100 mg/kg/day, gavage, 2 daily treatments at 24-hour intervals

Examinations

Tissues and cell types examined:

Two to six hours after the second treatment, the rats are killed and cells from the selected target organs (i.e. liver, glandular stomach and duodenum) are isolated for the comet assay.

Fives males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.
Single cell preparations were done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles was removed to reveal the abdominal cavity.
A portion of the liver, glandular stomach and duodenum was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Number of cells observed per animal : 150
Number of cells observed per dose : 750

Details of tissue and slide preparation:

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unwinding and electrophoresis in alkaline conditions and under dimmed light to prevent any additional DNA damage. After neutralization, slides are dried and could therefore be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows indeed the calculation of metrics for DNA migration.

Determination of the cytotoxicity of the test item :
In the main assay, the frequency of hedgehogs in the control group and in the 2000, 1120 and 410
mg/kg/day treated groups was inferior to 50%. Indeed maximal values were of 1.81%, 10.55% and 5.64% in the liver, the stomach and the duodenum, respectively and comparable to the values obtained in the respective control. The test item was thus considered as not cytotoxic.

Evaluation criteria:

Expression of the results
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of median per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Hedgehogs were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal, and per type of treatment and per organ. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis. A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.

A study is accepted if the following criteria are fulfilled:
- Concurrent vehicle controls should be within the control limits of the distribution of the laboratory’s historical vehicle control database.
- The concurrent positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent vehicle control.
- The appropriate number of doses and cells must be analysed.

Moreover:
- In the vehicle group, an eventual increase in the frequency of hedgehogs, must not be >50%.
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 %
per group. The dead animals from the high dose treated group are replaced by those in parallel treatment.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian
distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed.
This method is based on the analysis of variance by ranks for testing equality of population medians
among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the
vehicle control in order to determine statistical significance of differences in group median values
between each group versus the vehicle control. This test was also used to compare vehicle control
and positive control to determine acceptable criteria of a valid test.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

in glandular stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Sex:

male

Genotoxicity:

negative

Remarks:

in duodenum cells

Toxicity:

no effects

Vehicle controls validity:

not valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The animal demonstrated no clinical signs once dosed with the test item, whatever the dose tested.

Liver cells :
The value for the percentage of DNA in tail in the vehicle control was within the control limits of the distribution of the laboratory’s historical vehicle control database. Moreover, the concurrent positive control induced a response that was lower but comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the liver were fulfilled. The experiment is thus valid.
No statistically significant increase in the mean of medians of percentage of DNA in tail per slide was observed at the any tested doses from 410 to 2000 mg/kg b.w./day of TPS 32 in hepatocytes from OFA Sprague-Dawley male rats.
Otherwise, a statistically significant decrease in the mean of medians of percentage of DNA in tail per slide were noted in the group treated with 2000 mg/kg b.w./day of TPS 32 with a value of 0.03 vs. 0.14 in the respective vehicle control. The median of the percentage of DNA in tail was close to the lowest value already observed in historical data for vehicle control i.e. 0.02, and no dose effect relationship was noted. This change was therefore considered as not biologically relevant.
Overall, it is concluded that TPS 32 did not induce any DNA strand break in hepatocytes from OFA Sprague-Dawley male rats as investigated by the in vivo Comet assay.

Glandular stomach cells :
The value for the percentage of DNA in tail in the vehicle control was within the control limits of the distribution of the laboratory’s historical vehicle control database. Moreover, the concurrent positive control induced a response that was comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the glandular stomach were fulfilled. The experiment is thus valid.
No statistically significant increase in the mean of medians of percentage of DNA in tail per slide was observed at the any tested doses from 410 to 2000 mg/kg b.w./day of TPS 32 in glandular stomach from OFA Sprague-Dawley male rats.
Otherwise, statistically significant decreases in the mean of medians of percentage of tail DNA per slide were noted in the group treated with 1120 and 2000 mg/kg b.w./day of TPS 32 with values of 1.38 and 0.80, respectively vs. 5.04 in the respective vehicle control. The decrease was dose-related and the value for the mean of median of the percentage of DNA in tail at the top dose of 2000 mg/kg b.w./day was close to the lowest value already observed in historical data for vehicle control i.e. 0.80 vs. 0.74.
Overall, it is concluded that TPS 32 did not induce any DNA strand break in glandular stomach
from OFA Sprague-Dawley male rats as investigated by the in vivo Comet assay.

Duodenum :
The value for the percentage of DNA in tail in the vehicle control was outside the control limits of the distribution of the laboratory’s historical vehicle control database with a value of 0.16% vs. 0.21%. In return, the concurrent positive control induced a response that was comparable to the historical positive control data and produced a statistically significant increase compared with the concurrent vehicle control.
The appropriate number of doses (3) and cells (750) were analysed.
Moreover, in the vehicle group, no increase in the frequency of hedgehogs was noted.
The validity criteria for the experiment carried out in the duodenum was thus not completely fulfilled.
According to the OECD guideline 489, the data should be invalidated.
Scientifically, with a background of DNA fragmentation slightly lower than the minimal bound of the negative historical control, the sensitivity of the current assay could be considered as enhanced while a clear negative response was obtained meaning that the test item is not genotoxic in duodenum.

Analytical results :
The control of concentrations of TPS 32 in treatment preparations was performed in a GLP compliant laboratory following a validated method. The results are reliable.
The results of the assays for TPS 32 in treatment preparations were valid and considered as correct (only a slight deviation was noted at the lower dose). In addition, TPS 32 was not detected in the solvent control.

Applicant's summary and conclusion

Conclusions:

Overall, under our experimental conditions, the test item does not present DNA strand breaks
and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA
Sprague-Dawley male rats.
No definitive conclusion can be drawn for duodenum. For the duodenum, median of
percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound
of negative historical control data. If it constitutes a deviation according to OECD guidelines
489 and doesn’t fulfil the acceptance criteria of the results, the clear negative response
observed in this organ tends to confirm the lack of genotoxicity of TPS32 in duodenum.

Executive summary:

The search for potential genotoxic activity of TPS 32 (Polysulfides, di-tert-dodecyl - CAS 68425-15-0) was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat.
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the maximum recommended dose, i.e. 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and glandular stomach. Noteworthy, significant and dose-related decreases in the percentage of tail DNA were noted in glandular stomach but the values are included or close to the historical control data.
Overall, under our experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and glandular stomach from OFA Sprague-Dawley male rats.
No definitive conclusion can be drawn for duodenum. For the duodenum, median of percentage of tail DNA in the vehicle control group was slightly lower than the minimal bound of negative historical control data. If it constitutes a deviation according to OECD guidelines 489 and doesn’t fulfil the acceptance criteria of the results, the clear negative response observed in this organ tends to confirm the lack of genotoxicity of TPS32 in duodenum.