Registration Dossier

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:
22 June 2020 to 03 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(triethoxysilyl)propanethiol
EC Number:
238-883-1
EC Name:
3-(triethoxysilyl)propanethiol
Cas Number:
14814-09-6
Molecular formula:
C9H22O3SSi
IUPAC Name:
3-(triethoxysilyl)propanethiol
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: W I(Han)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sex: male animals were used in the main study as no differences were observed between the male and female animals used in the preliminary dose range-finding study.
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at dosing in main experiment: 253-304 g
- Assigned to test groups randomly: yes. Prior to randomization detailed clinical observations were made to assure a good health condition. Animals showing pathological signs before administration were excluded from the study. Supplementary animals from the same delivery were provided in exchange.
- Fasting period before study: no
- Housing: The animals were kept in groups of 2-3 animals / sex / group / cage in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum. (Altromin Spezialfutter GmbH & Co. KG Im Seelenkamp 20, D-32791 Lage)
- Water tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals, ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not given in study report

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: none
- Justification for choice of solvent/vehicle: the substance hydrolyses rapidly in contact with water, so no vehicle was used
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was administered undiluted.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Daily
Post exposure period:
Animals were sacrificed 4 hours after second administration of test substance
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Five
Control animals:
yes
Positive control(s):
- Positive control substance: ethylmethanesulfonate;
- Justification for choice of positive control(s): none given in report. This is a standard positive control substance for the in vivo mammalian alkaline comet assay
- Route of administration: oral
- Doses / concentrations: 200 mg/kg bw, single dose

Examinations

Tissues and cell types examined:
Cells of the liver, glandular stomach and duodenum were examined
Details of tissue and slide preparation:
In vivo phase (BSL Bioservice)
CRITERIA FOR DOSE SELECTION: The highest dose was based on the maximum tolerated dose, defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, determined in a dose range-finding study. In this preliminary study, 2 male and 2 female animals were dosed with the limit dose of 2000 mg/kg bw daily for 2 consecutive days. Clinical signs were observed in all animals, and the male and female rats were euthanised 4 hours after the second treatment due to severity of symptoms and out of consideration of animal welfare. Based on these effects, a dose of 500 mg/kg bw was administered orally on 2 consecutive days with a 24-hour interval to one male and one female animal. After the second application adequate toxicity was not observed at this dose, and a further two animals (one male, one female) were dosed orally with 1000 mg/kg bw on two consecutive days. Adequate toxicity was observed at this dose level, and no gender specific differences were determined. Based on these results, the main study was conducted with male rats and 1000 mg/kg bw, which is the limit dose according to the OECD test guideline 489, was selected as the highest dose in the main experiment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Treatment occurred during two consecutive days. Animals were sacrificed 4 hours after the second dose. Sampling occurred after termination of the animals.

Tissue Sampling
Four hours after last treatment the liver, glandular stomach and duodenum were removed and a part of each was kept in ice-cold mincing buffer for the comet assay. The other part of isolated organs was preserved in 10% neutral-buffered formalin for histopathological evaluation, if considered necessary by the sponsor.
Organs assigned to comet assay were rinsed with cold mincing buffer to remove residual blood and stored in mincing buffer on ice until further processing. The times between animal death and removal of the tissues as well as the times to process the tissues and slides were tightly controlled and recorded in the raw data.

Ex vivo phase
Tissue preparation:
Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of duodenum cells:
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. Afterwards a portion of the duodenum was minced with a pair of scissors. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

Isolation of glandular stomach cells:
The glandular stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.

DETAILS OF SLIDE PREPARATION:
The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).

Lysis:
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.
Unwinding of DNA and electrophoresis:
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.
Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralized by rinsing with neutralization buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.
DNA staining:
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet may be adjusted manually if they were not set correctly automatically. All cells of the visual field were scored, except for e.g. overlapping cells, cells with an atypical nucleus, cells with a strong background or “hedgehogs” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail, are considered to be heavily damaged cells). Therefore, cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells).
To avoid artefacts, only scorable cells (defined round to oval nucleus) and at least 150 cells per sample on two slides (75 cells per slide) were scored. The third back-up slide was scored in case of discordant results. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus.
Evaluation criteria:
The assay is considered acceptable if:
- the concurrent negative control data are considered acceptable for addition to the laboratory historical control database,
- the concurrent positive controls should induce responses that are compatible to those previously generated and included in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control,
- three doses and, if available, 150 cells per organ of each animal have been analysed.

A test item is considered to be clearly positive if:
- acceptability criteria are fulfilled
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data
A test item is considered clearly negative if:
- acceptability criteria are fulfilled
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).
Statistics:
All slides, including those of positive and negative controls were independently coded and blinded before microscopic analysis. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was then determined to give a group mean % of tail DNA. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
some signs of systemic toxicity were observed in the high dose animals
Vehicle controls validity:
not applicable
Remarks:
No vehicle was used; the negative control, 0.9% NaCl, was valid.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1000 and 500 mg/kg bw
- Solubility: not relevant
- Clinical signs of toxicity in test animals:
2000 mg/kg bw: Clinical signs were observed in all animals, and the male and femal rats were euthanised 4 hours after the second treatment due to severity of symptoms and out of consideration of animal welfare.
500 mg/kg bw: after the second application the only clinical sign left was moving bedding in both genders, which had resolved after 30 minutes.
1000 mg/kg bw: after the second application reduced spontaneous activity, piloerection, moving bedding and salivation, prone position, ataxia and chromodacryorrhea were present. 4 hours later most of the animals recovered from the symptoms. No gender specific differences were determined. Based on these results, the main study was conducted with male rats and 1000 mg/kg bw, which is the limit dose according to the OECD test guideline 489, was selected as the highest dose in the main experiment.
- Evidence of cytotoxicity in tissue analysed: not applicable
- Rationale for exposure: initial limit dose showed excessive toxicity, the second dose was too low to elicit adequate toxicity, so a third dose was administered


RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: both route of exposure and dose were appropriate
- Results and statistical evaluation: no statistically significant increase in the mean tail intensity relative to the negative control was observed in liver, duodenum and glandular stomach cells of male animals treated with the test substance.

Any other information on results incl. tables

Summary of Mean Tail intensities [%] in liver, glandular stomach and duodenum

Dose

 

Mean Tail Intensity

[%]

 

Group

Liver

Glandular stomach

Duodenum

Clinical Signs

PC

11.83*

16.86*

11.36*

None

NC

1.62

1.88

1.79

None

LD

1.69

2.68

4.16

Moving bedding, salivation

MD

2.01

2.94

3.73

Moving bedding, salivation

HD

0.68

4.44

2.35

e.g. reduced spontaneous activity, prone position, half eyelid closure, piloerection

Statistically significant trend

Yes**

No

No

/

Historic Control Range

Negative Control: 0.00° % – 6.11 %

Positive Control: 3.14 % – 31.43 %

Negative Control:

0.89 % – 8.73%

Positive Control: 4.66% - 31.23%

Negative Control:

0.00° % – 7.47%

Positive Control: 3.89% - 33.14%

/

*      significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

**     statistically significant concentration-dependency was noted but not considered biologically relevant

°      A negative value was obtained due to the calculation of the LCL. However, as no negative tail intensities can be measured, the LCL was set to 0.

 

NC: Negative Control (0.9% NaCl)

LD: Low Dose

PC: Positive Control (Ethyl methanesulfonate: 200 mg/kg bw)

MD: Mid dose

HD: High Dose

n.a.: Not available

Applicant's summary and conclusion

Conclusions:
In an in vivo mammalian alkaline comet assay conducted in accordance with OECD test guideline 489 and in compliance with GLP, 3-(triethoxysilyl)propanethiol was administered by oral gavage to groups of five male Wistar rats at doses of 250, 500 and 1000 mg/kg bw daily for two consecutive days. The test substance did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum. The results of the negative control were within laboratory historical control values, and the positive control substance induced a statistically significant increase in tail intensity; the validity criteria were therefore met. It is concluded that 3-(triethoxysilyl)propanethiol is negative for the induction of DNA damage in the in vivo mammalian alkaline comet assay, under the conditions of the experiment.
Executive summary:

3-(Triethoxysilyl)propanethiol has been tested in a reliable in vivo mammalian alkaline comet assay conducted according to OECD Test Guideline 489 and in compliance with GLP. In an initial dose range-finding study toxicity was observed but there were no relevant differences between male and female animals. Based on the results of the preliminary study, the test substance was administered to male Wistar rats by oral gavage at doses of 250, 500 and 1000 mg/kg bw. The animals were dosed twice over two days. 0.9% sodium chloride was used as a negative control; the positive control was ethyl methanesulfonate, administered once by oral gavage at a dose of 200 mg/kg bw.

No signs of systemic toxicity were observed in the low and mid dose groups, except moving bedding and slight salivation in two animals per group. The animals treated with the 1000 mg/kg bw showed moving bedding, salivation, reduced spontaneous activity, prone position, piloerection, hunched posture and half eyelid closure.

The animals were sacrificed four hours after the last treatment, and cells from the liver, the glandular stomach and the duodenum were isolated, embedded in agarose, lysed and DNA allowed migrating under electrophoresis conditions. 150 cells per animal tissue were evaluated. DNA migration during electrophoresis was determined and expressed as tail intensity.

The tail intensities of the negative control group were within the historical control limits, and the mean values for the test groups were within the range of the concurrent negative control. The positive control induced a statistically significant increase in DNA damage for all evaluated organs.

In conclusion, under the experimental conditions reported in this study, the test item 3‑(triethoxysilyl)propanethiol did not induce biologically relevant DNA-strand breaks in liver, glandular stomach and duodenum after oral administration to rats.

Therefore, 3-(triethoxysilyl)propanethiol is considered to be negative for the induction of DNA damage under these experimental conditions in the in vivo mammalian alkaline comet assay.