Registration Dossier

Administrative data

in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020/11/23 - 2021/03/23
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
3-(triethoxysilyl)propyl thiocyanate
Test material form:

Test animals

Crl: WI(Han)
Details on test animals or test system and environmental conditions:
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: Approximately 7-9 weeks
- Weight at study initiation: 164-199 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: In groups of 2-3 animals / sex / group / cage, in IVC cages (type III H, polysulphone cages)
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: Tap water, sulphur acidified to a pH of approximately 2.8, ad libitum
- Acclimation period: At least 5 days

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

Administration / exposure

Route of administration:
oral: gavage
None, test item administered undiluted
Details on exposure:
Triethoxy(3-thiocyanatopropyl)silane was administered undiluted at 25, 50, 75 mg/kg bw/day. The administered volume of the test item was adjusted to the animal’s body weight by dose group:
- 0 mg/kg bw/day: 2 mL/kg bw
- 25 mg/kg bw/day: 0.0315 mL/kg bw
- 50 mg/kg bw/day: 0.0630 mL/kg bw
- 75 mg/kg bw/day: 0.0950 mL/kg bw
- ethyl methanesulfonate (positive control): 10 mL/kg bw
Duration of treatment / exposure:
Over 2 days
Frequency of treatment:
Once a day for two consecutive days
Post exposure period:
None, animals were sacrificed on day 2 (4 hr after dosing)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
animals received 0.9% NaCl as negative control
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
maximum tolerated dose in range-finding study
No. of animals per sex per dose:
Control animals:
Positive control(s):
yes, ethyl methanesulfonate at 230 mg/kg bw, administered 4 hr before sacrifice


Tissues and cell types examined:
The liver, glandular stomach and duodenum were examined. The organs were selected to cover two first-contact organs upon peroral exposure and liver as the metabolising organ.
Details of tissue and slide preparation:
Four hours after the last treatment animals were deeply anaesthetized using ketamine/xylazine. The abdominal aorta was cut and the blood was released. 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 the isolated organs was preserved in 10% neutral-buffered formalin for histopathological evaluation, if considered necessary by the sponsor.

Organs assigned to the 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.

The 25 samples of liver, 25 samples of glandular stomach and 25 samples of duodenum were analysed for DNA strand breaks. The samples were kept in ice-cold mincing buffer. The fresh samples were directly prepared according to the following procedure using a small part of the available tissues. The remaining part of all tissues was fixed for enabling a possible histopathological analysis.

Isolation of primary hepatocytes:
A portion of the liver was minced with a pair of scissors to liberate the cells. The cell suspension was left 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 low-melting agarose (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 left 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 left 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.

In accordance with OECD Test Guideline 489, 150 cells/animal/tissue were analysed, if available.

Once single cells were obtained, the basic steps of the assay included:

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).

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 are incubated in alkaline (pH > 13) electrophoresis solution for 20 min. An incubation period of 20 min is generally considered appropriate for alkali unwinding.

After alkali unwinding, the unwound DNA is electrophoresed under alkaline conditions to allow for the formation of DNA tails. The electrophoretic conditions are 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min. The slides are placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis buffer. Slides will be placed in the electrophoresis chamber in a random order.

Neutralization 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.

Analysis of DNA-strand breaks:
Comet slides are analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and by using the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). The slides will be coded so that the evaluator is not aware of which dose group is evaluated. Each slide is screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters will be done automatically by the Comet Software, but the set front, middle and back lines of the comet may be adjusted manually if they are not correctly set automatically. All cells of the visual field will be 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 will be classified into three categories: scorable, non-scorable and “hedgehog”. 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) should be scored. The third back-up slide may be scored in case of discordant results. The %-tail intensity will be the parameter for evaluation and interpretation of DNA damage, and is 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:
Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.

Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- 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

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- 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).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.
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 negative control.

Results and discussion

Test results
Key result
No biologically relevant increase of tail intensity was found after treatment with triethoxy(3-thiocyanatopropyl)silane in any of the dose groups and organs evaluated compared to the negative (0.9% NaCl) controls.
At 75 mg/kg bw/day. No signs systemic toxicity at 25 or 50 mg/kg bw/day.
Vehicle controls validity:
Tail intensities of the negative (0.9% NaCl) control group were within the historical control limits and, therefore, accepted for addition to the laboratory control data set.
Negative controls validity:
See "vehicle controls validity"
Positive controls validity:
The positive control ethyl methanesulfonate induced a statistically significant increase in DNA damage for all evaluated organs.
Remarks on result:
other: See Table 1 below
Additional information on results:
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

- Appropriateness of dose levels and route:
- Other: At 75 mg/kg bw/day, animals showed mild to moderate toxic effects such as moving bedding, salivation, reduced spontaneous activity, half eyelid closure, piloerection and abnormal breathing. No signs systemic toxicity at 25 or 50 mg/kg bw. The body weight variation in the main experiment (±9.7%) was below 20%.

Any other information on results incl. tables

Table 1. Summary of mean tail intensities [%] in liver, glandular stomach and duodenum



Mean tail intensity [%]

Clinical signs


Glandular stomach


25 mg/kg bw/d





50 mg/kg bw/d





75 mg/kg bw/d




e.g. Moving bedding, salivation,reduced spontaneous activity, prone position

Statistically significant trend





Study positive control (a)





Study negative (b) Control





Historic control range

Negative Control: 0.07 % – 3.82 %

Positive Control:

2.81 % – 28.08 %

Negative Control:

1.30 % – 5.46%

Positive Control: 5.99% - 34.62%

Negative Control:

0.89 % – 4.06%

Positive Control: 1.77% - 30.75%


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

(a) ethylmethanesulfonate, 200 mg/kg bw

(b) 0.9% NaCl

Applicant's summary and conclusion

In a study conducted according to OECD Test Guideline 489 and in compliance with GLP (reliability score 1), triethoxy(3-thiocyanatopropyl)silane did not induce biologically relevant DNA-strand breaks in the liver, glandular stomach and duodenum of female Wistar Han rats. Rats were administered undiluted triethoxy(3-thiocyanatopropyl)silane at 25, 50, and 75 mg/kg bw/day via oral gavage. None of the test substance-treated animal slides had significant increases in the % tail DNA compared to the respective negative control (0.9% NaCl). All criteria for a valid assay (0.9% NaCl negative control, ethyl methanesulfonate positive control) were met for the liver, glandular stomach and duodenum. Under the experimental conditions, triethoxy(3-thiocyanatopropyl)silane was not DNA-damaging.