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:
13 May - 24 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
adopted Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (Chloromethyl)triethoxysilane
- Physical state: colourless liquid
- Density: 1.048 g/ml (at 20 °C, 1013 hPa)
- Lot/batch No.: KH09731
- Expiration date of the lot/batch: 01 August 2020
- Storage condition of test material: at room temperature (20±5 °C) and protected from light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 7-9 weeks old
- Weight at study initiation: interval within ± 20 % of the mean weight, if technically possible
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: group housing of 5 animals / sex / group / cage in IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap-water, ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- 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
Vehicle:
- Vehicle used: corn oil
Details on exposure:
the test item was formulated in corn oil at a concentration of 200 mg/mL just before administration and diluted prior to treatment.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once daily
Post exposure period:
4 hours after last treatment
Doses / concentrationsopen allclose all
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethylmethanesulphonate
- Route of administration: oral
- Doses / concentrations: 10 mL/kg bw; 200 mg/kg bw

Examinations

Tissues and cell types examined:
liver, glandular stomach and duodenum
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels for the main experiment were chosen based on the results from the pre-experiment for toxicity.

TREATMENT AND SAMPLING TIMES:
The test item and vehicle control item were administered orally via gavage. Prior to the administration, food was not withheld. The application volume for the groups was 10 mL/kg bw. Treatment of groups was performed daily and the last treatment was on day 2 4 hours before animal sacrifice.
The positive control item was administered only once 4 hours before animal sacrifice with a dose of 200 mg/kg bw EMS and an application volume of 10 mL/kg bw.

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 was then 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 for the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed with 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. An incubation period of 20 min is generally considered appropriate for alkali unwinding.
After alkali unwinding, the single-stranded DNA is 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 20 min. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis buffer. Slides were placed in the electrophoresis chamber in a random order.
Neutralisation and dehydration of slides:
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min. 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 solution on top of the slides and covering with a cover slip.


METHOD OF ANALYSIS:
Comet slides were analyzed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software “Comet Assay IV” (Perceptive Instruments, Software version 2.1.2). The slides were coded so that the evaluator is not aware of which dose group was evaluated. Each slide was screened for cells in a meandering pattern in the unfrosted area of the slide by an evaluator. The calculation of the different parameters was done automatically by the Comet Software, but the set front, middle and back lines of the comet was adjusted manually if they are not set correctly automatically. All cells of the visual field was scored, except of 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, were considered to be heavily damaged cells). Therefore, cells were 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) were scored. The third back-up slides were scored in case of discordant results. The %-tail intensity was 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

OTHER: All animals were observed for clinical signs during the entire treatment period of 2 days. General clinical observations related to the health of the animals were made and recorded at least once a day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Body weight was recorded from all animals on each day of administration.

Evaluation criteria:
The alkaline comet assay is considered acceptable if it meets the following criteria according to OECD guideline 489:
- the concurrent vehicle control data are considered acceptable for addition to the laboratory historical control database,
- the concurrent positive controls should induce responses that are comparable 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.

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).
Statistics:
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 determined to give a group mean % of tail DNA.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
reduced body weight in highest dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In the pre-experiment, one female and one male rat received a single dose of 2000 mg/kg bw orally and showed severe signs of toxicity such as reduced spontaneous activity, prone position, moving the bedding, loss of weight, piloerection, ataxia and half eyelid closure (male only) eyelid closure (female only). After second application of the test item animals were euthanized due to the animal welfare reasons. Therefore, the dose was reduced to 1000 mg/kg bw. One male and one female rat showed sights of toxicity such as reduction of spontaneous activity, piloerection, wasp waist (females only), prone position (females only) and significant reduction of body weight.
Due to the sights of toxicity the dose of test item was further reduced to 700 mg/kg bw. Three males and three females were treated twice on two consecutive days, with test item and the following mild clinical symptoms were obtained: reduction of spontaneous activity (males only), piloerection and diarrhea (males only). There was only a slight reduction in body weight.
- Clinical signs of toxicity in test animals: A reduction of body weight was noted in each male animal in HD group (700 mg/kg bw) at the end of the experiment wheras the other test groups gained weight.
- Harvest times: 4 hours after second treatment

Any other information on results incl. tables

  1. Tail intensity in Liver Cells

The tail intensity of the LD (1.09 %), MD (0.97 %) and HD (1.20 %) were within the historic control limits (0.09 – 6.44 %).

Tail Intensity [%] in                   

Liver Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

7.41

0.55

0.51

1.28

0.88

II

8.87

1.03

1.23

1.39

0.95

III

5.57

0.88

0.76

0.67

0.58

IV

10.91

1.02

1.44

1.13

1.56

V

7.48

2.08

1.54

0.38

2.03

Group Mean:

8.05

1.11

1.09

0.97

1.20

± SD:

1.98

0.58

0.44

0.43

0.59

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

  1. Tail intensity in Glandular Stomach Cells

The tail intensity of the LD (1.71 %), MD (2.86 %) and HD (2.96 %) were within the historic control limits (1.76 – 9.24 %).

Tail Intensity [%] in                    

Grandular Stomach Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

9.07

1.61

1.20

2.45

2.84

II

10.07

1.92

1.25

4.10

4.72

III

7.08

1.81

2.23

3.28

2.53

IV

13.71

1.78

2.82

1.61

2.58

V

13.68

2.75

1.07

2.86

2.13

Group Mean:

10.72

1.97

1.71

2.86

2.96

± SD:

2.92

0.44

0.77

0.93

1.02

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

  1. Tail intensity in Duodenum Cells

The tail intensity of the LD (1.56 %), MD (1.22 %) and HD (1.71 %) were within the historic control limits (0.00 -8.13%).

Tail Intensity [%] in                   

Duodenum Cells

Animal per Group

Mean of Medians

PC

VC

LD

MD

HD

I

9.34

1.14

1.14

1.97

1.60

II

15.81

1.00

1.53

0.78

1.90

III

9.75

1.92

2.14

1.24

1.39

IV

11.03

0.46

1.74

1.53

1.39

V

17.37

1.04

1.24

0.58

2.28

Group Mean:

12.66

1.11

1.56

1.22

1.71

± SD:

3.68

0.52

0.40

0.56

0.38

 

 

 

 

 

 

 

HD:

High Dose

LD:

Low Dose

MD:

Mid Dose

PC:

Positive Control (Ethyl methansulfonate: 200 mg/kg bw)

SD:

Standard Deviation

VC:

Vehicle Control (Corn Oil)

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative