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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Comet Assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11th of June 2019 to 31th of July 2019
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:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29th of July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
W1 (Han) rats (SPF)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: For males, 155 ± 4.9 g. Unspecified initiation weight for females.
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food (appr. 7 g/rat) was supplied during the night before dosing.
- Housing: Maximum 5 animals per sex in Macrolon cages (type MIV: 180*600*3300 mm, containing sterilised sawdust as bedding material and paper as cage-enrichment.
- Diet: Ad libitum to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany).
- Water: Ad libitium
- Acclimation period: At least 5 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 20-21°C
- Humidity: 49-73%
- Air changes: =10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 12th of June 2019 to 18th of July 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Dried corn oil
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: Specific gravity of dried corn oil was 0.9 g/ml
- Amount of vehicle: not specified
- Type and concentration of dispersant aid: n/a
- Lot/batch no.: n/a
- Purity: not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved (clear yellow) in dried corn oil.
Duration of treatment / exposure:
2 days
Frequency of treatment:
Once a day for 2 days
Post exposure period:
Animals were sacrificed 3-4 hours after the second dose
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
solvent control
Dose / conc.:
500 mg/kg bw/day
Remarks:
test substance
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
test substance
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
test substance
Dose / conc.:
200 mg/kg bw/day
Remarks:
positive control
No. of animals per sex per dose:
Main study: 5 males per dose. Dose range finding study: one group of 3 male and 3 female animals.
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
Positive control: Ethyl methanesulfonate (EMS)
- Justification for choice of positive control(s): not specified
- Route of administration: oral
- Doses / concentrations: 10 mL/kg bw/day
- Other: EMS at 200 mg/kg bw/day was dissolved in physiological saline

Examinations

Tissues and cell types examined:
Liver, duodenum and glandular stomach cells were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A dose range finding study was performed prior to this experiment where suitable doses were selected. In total 3 male and 3 female animals were dosed with the limit dose of 2000 mg/kg bw for 2 consecutive days. Clinical signs were recorded 2 hours and 21 hours after each dose. No mortalities occurred and no abnormalities were observed, therefore 2000 mg/kg bw was selected as the maximum dose.

TREATMENT AND SAMPLING TIMES: Treatment occurred during two consecutive days. Animals were sacrificed 3-4 hours after the second dose. Sampling occurred after termination of the animals.

DETAILS OF SLIDE PREPARATION:
Preparation of slides: Melted agarose was added to each cell suspension and was layered on a pre-coated Comet slide in duplicate. Subsequently, the slides were incubated for 10-11 minutes in a dark refrigerator until a clear ring appeared on the edge of the Comet slide area. Three slides per tissue per animal were prepared.

Lysis, electrophoresis and staining of the slides: Slides with the cells were immersed overnight in lysis solution in a refrigerator. Following the immersion overnight, the slides were rinsed with neutralisation buffer and placed in an alkaline solution at room temperature in the dark. Thereafter, the slides were placed in an electrophoresis unit with the applied voltage of 0.7 Volt/cm for 20 minutes for duodenum and glandular stomach slides or 30 minutes for liver slides. Following rinsing in neutralisation buffer and immersing in absolute ethanol, the slides were dried at room temperature. A fluorescent dye was utilised to stain the slides which was washed away after a few minutes and the slides were leaved to dry at room temperature in the dark and fixed with a coverslip.

METHOD OF ANALYSIS: Prior to the examination of Comets, slides were randomly coded by adding an adhesive label per tissue which also prevented bias occurring. Thereafter, a fluorescence microscope connected to a Comet Assay IV image analysis system was utilised to examine the slides. 150 comets were examined per sample where the following criteria for scoring of comets were used:
• Only horizontal orientated comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.


HISTOPATHOLOGY:
Preparation of histopathology: Collection of parts of the liver, duodenum and glandular stomach were fixed in buffered formalin until further use. It was evidenced that the tail intensity was significantly increased in the animals dosed with 2000, 1000 and 500 mg/kg bw/day, which is why histopathology of these doses and the vehicle was further examined. For histopathology purposes, the tissues were fixed in paraffin wax, cut to 2-4 µm and thereafter stained with haematoxylin and eosin.
Evaluation criteria:
The in vivo Comet Assay was considered acceptable if it met the following criteria:
a) The concurrent negative control data were considered acceptable when they were within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle-treated animals. The response should be compatible with the data in the historical control database. In case of homogeneous or inhomogeneous variances, the positive control data was analysed by the Students t test or by the Welch t-test.
c) Adequate numbers of cells and doses had been analysed
d) The highest test dose was the MTD or 2000 mg/kg body weight/day
A test item is considered positive in the Comet Assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity detected compared with the concurrent negative control.
b) The increase is dose-related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
Statistics:
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data. The data showed inhomogeneous variances and therefore the Welch t test with Bonferonni-Holm Adjustment was applied. A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
positive
Remarks:
A statistically significant increase of the mean tail intensity of cells from the glandular stomach, duodenum and liver at all doses tested (which were above the historic control limit) was observed.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw for two consecutive days
- Solubility: no information
- Clinical signs of toxicity in test animals: no effects observed.
- Evidence of cytotoxicity in tissue analysed: not applicable
- Rationale for exposure: limit dose

RESULTS OF DEFINITIVE STUDY
- Result and Statistical evaluation: A statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of male animals treated with the test item compared to the vehicle-treated animals. All the treatment groups showed Tail Intensity increases above the historical control data of the vehicle-treated animals and in all organs this effect showed a dose-related increase shown by a significant trend analysis.
- Appropriateness of dose levels and route: The route of exposure and dose levels were appropriate according to the guideline.
- Necropsy and histopathology: No gross lesions were observed at necropsy. No histological alterations were noted in any specimen from any group.

No cytotoxicity in the target tissues and no clinical effects were observed in the treated animals. Possible test item-related morphologic alterations (aggravation of eosinophilic cell infiltrates in the submucosa) were present in the glandular stomach at 2000 mg/kg bw.

Applicant's summary and conclusion

Conclusions:
3-(2,3-Epoxypropoxy)propyltrimethoxysilane has been tested in a valid in vivo mammalian alkaline comet assay study, conducted according to OECD TG 489 and in compliance with GLP. Statistically significant increases in the mean tail intensity of cells from the glandular stomach, duodenum and liver was reported at all doses tested.
Executive summary:

3-(2,3-Epoxypropoxy)propyltrimethoxysilane has been tested in a valid in vivo mammalian alkaline comet assay study, conducted according to OECD TG 489 and in compliance with GLP. Groups of 5 male Wistar WI (Han) rats (SPF) were dosed via oral gavage over two consecutive days at doses of 500, 1000 and 2000 mg/kg bw. No cytotoxicity was reported in the target tissues and no clinical effects were observed in the treated animals. Possible test item-related morphologic alterations (aggravation of eosinophilic cell infiltrates in the submucosa) were present in the glandular stomach at 2000 mg/kg bw. Statistically significant increases in the mean tail intensity of cells from the glandular stomach, duodenum and liver was reported at all doses tested.