Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): positive with and without activation in TA97 and TA100 (similar to OECD Test Guideline 471) (Microtest Research Ltd., 1988).
Mutagenicity in mammalian cells: positive in L5178Y mouse lymphoma L5178Ycells (similar to OECD Test Guideline 476) (Litton Bionetics, 1983); negative in Chinese hamster Ovary (CHO) cells (similar to OECD Test Guideline 476, Reliability 4), (Allied Corporation, 1979).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-09-09 to 1987-10-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were that only three strains of bacteria were used.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only three strains used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
8, 40, 200, 1000, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether

- Justification for choice of solvent/vehicle: none given in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 97 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium Azide
Remarks:
TA 100 (without activation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains (with activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Expression time (cells in growth medium): 48 hours

NUMBER OF REPLICATIONS: 3 plates per test concentration

DETERMINATION OF CYTOTOXICITY

- Method: relative total growth; background lawn assessment
Evaluation criteria:
Colony counts for test substance compared with solvent controls. A positive response is indicated by a two-fold increase in mean revertant numbers compared with the solvent control.
Statistics:
Mean values calculated from individual plate counts. Statistical significance calculated using F-test.
Key result
Species / strain:
S. typhimurium, other: TA97 and TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A statistically significant increase in revertants noted in Salmonella typhimurium strains TA 97 and TA 100 both in the absence and presence of S-9.

Table 2 :  Mutagenicity assay, Number of revertants per plate (mean of 3 plates)

 

TA 97

TA 98

TA 100

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

— MA

+ MA

Cytotoxic
(yes/no)

0*

77.8

142.2

No

18.2

43.4

No

87.4

133.6

No

8

62

123.7

No

14

46.3

No

96

141

No

40

63.3

132

No

18

34.5

No

98.7

144.7

No

200

57.3

135.7

No

22

35.3

No

120

169.7

No

1000

70.7

168.3

No

19.3

34.3

No

243

301.3

No

5000

154.7

276.7

No

18.7

44.3

No

598.7

690.7

No

Positive Control

340.8

357.8

No

905

969.6

No

502.8

432.2

No

*solvent control with Ethylene glycol dimethylether

Conclusions:
3-glycidoxypropyltrimethoxysilane (CAS 2530-83-8) (GLYMO) has been tested in a valid study according to a protocol that is similar to OECD TG 471 and under GLP. GLYMO produced a fivefold increase in revertants in Salmonella typhimurium TA 100, and a two fold increase in TA 97 with and without metabolic activation. The test substance is considered mutagenic in Salmonella typhimurium strains TA97 and TA100 both in the absence and presence of S-9 under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-03-10 to 1983 --05-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
With and without activation assays were performed separately. Only a generic method was provided.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Method: other
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 including NADP as cofactor
Test concentrations with justification for top dose:
100, 300, 500, 1000, 2000, 3000 nl/ml (non-activation), 2000, 4000, 5000, 6000, 8000 nl/ml (with S9 activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The report states "the test material was prepared as specified by the client, just prior to each assay.
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: none
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2-3 days

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: duplicate cultures, 3 plates per culture for mutant frequency derivation. The with activation assay was repeated twice as the first experiment activation solvent and untreated control had an unacceptable high background mutant frequency.

NUMBER OF CELLS EVALUATED: 3 x 10 E+06

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:
Not given in report.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3.13 nl/ml without activation and 12.5 nl/ml with activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was probably increased at 12.5 µl/ml and above, as indicated by pink colour probably resulting from phenol red indicator in media.
- Effects of osmolality: no data
- Precipitation: noted at 6.25 to 100 µl/ml

Appropriate concurrent negative and positive controls were
included, and the expected responses were observed.

Under non-activation conditions, a dose-dependent increase
in mutant frequency was induced and exceeded the minimum
criteria for mutagenicity (83.4 X 10-6) at 2000 nl/ml and
3000 nl/ml.  Low to moderate toxicity was induced (percent
relative growth 97% - 37.7%).  

In the presence of metabolic activation a wide range of
toxicities was observed (percent relative growth 91.2% -
8.9%).  All the assayed treatments exceeded minimum
criterion of 68.0 X 10-6.  However, relative growth was
dramatically inhibited at concentrations of 5000 nl/ml or
greater.

 Table Results of mutagenicity testing

Concentrations

nl/ml

Expt 1 without activation

Expt 2 and 3 with activation

Relative growth (%)

Mutant frequency

Dose

nl/ml

Relative growth (%)

Mutant frequency

Solvent control (av of 2expt 1, of 3 expt 2 and 3)

100

45.9

-

100

46.7

Untreated control

121

65

-

119

32.1

Positive control low dose

55.3

348.7

-

92.5

238.9

Positive control high dose

92.3

447.8

-

52.8

-

100

123.1

33.7

2000

126.3

164.1

300

142.8

37.9

4000

59.0

289.6

500

96.1

51

5000

40.7

383.9

1000

93.9

53.3

5000

31.1

360.6

2000

92.1

103.9

6000

32.7

292.7

3000

65.3

151.3

6000

49.7

359.5

 

 

 

8000

17.6

388.1

 

Positive control low dose: 0.25 µl/ml EMS without activation, 0.15 µl/ml DMN with activation

Positive control 0.4 µl/ ml EMS without activation, 0.130 µl/ml DMN with activation

Conclusions:
3-Glycidoxypropyltrimethoxysilane (CAS No. 2530-83-8) has been tested in a valid study according to a protocol similar to OECD TG 476 and under GLP. The substance induced mutations in mouse lymphoma L1578Y TK cells, both with and without metabolic activation, in a dose dependent manner. It is considered that 3-Glycidoxypropyltrimethoxysilane is positive for the induction of mutations in mammalian cells under the conditions of this test.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 487
GLP compliance:
not specified
Remarks:
published journal without any statement whether GLP compliance was met
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mammalian cell line, other: HepG2 cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
other: human cell line from the European Collection of Cell Culture, UK.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
with metabolic activation: 0.03, 0.06, 0.125, 0.25, 0.5 µg/ml; without metabolic activation: 0.03, 0.06, 0.125, 0.25 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Vinblastine 0.625 ng/ml
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
10 µg with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

ACTIVATION: Phenobarbital/beta-naphthoflavone induced rat liver S9. S9 mix: final concentration in medium 2% S9; co-factors 5mM glucose-6-phosphate, 0.3 mM NADP, 1.5 mM KCl.

DURATION
- Exposure duration: 3 h with metabolic activation, 24 h without metabolic activation
- Expression time (cells in growth medium): 44 h in medium containing cytochalasin (4 µg/ml). 1 h before harvest cells were washed and re-fed MEM

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin

NUMBER OF REPLICATIONS: duplicate treatments

NUMBER OF CELLS EVALUATED: Micronucleus frequency determined in at least 2000 cells (at least 1000 from each culture). Micronuclei criteria: diameter < one third of main nucleus; clearly distinguishable from main nucleus and staining identical to main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: other: cytokinesis-block proliferation index (CBPI). CBPI =[(the number of cells with 1 nucleus x 1)+(the number of cells with greater than 2 nuclei x 3)]/total number of cells scored

Evaluation criteria:
According to OECD487
Statistics:
One-way ANOVA followed by Student-Newman-Keuls test, and the differences were considered significant for p less than 0.05.
Species / strain:
mammalian cell line, other: HepG2 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
50 % cytotoxicity at highest concentration in absence of metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

The results in the paper are presented in the form of bar charts. The numbers in the table below have been estimated from the charts.

Table 1 Results of micronucleus assay for GPTES 

Concentration

MN/1000 BNC

Cytotoxicity (%)

-MA

+MA

-MA

+MA

Solvent control

12

10

0

0

0.03

13

13

-6

0

0.06

11

13

-10

0

0.125

14

14

-3

0

0.25

12

12

50

2%

0.5

NT

11

NT

25%

Positive control

55

33

4

0

NT not tested

MN micronuclei

BNC binucleated cells

Conclusions:
[3-(2,3-epoxypropoxy)propyl]triethoxysilane has been tested for ability to induce formation of micronuclei in a study conducted according to OECD 478. No evidence of test-substance induced micronucleus formation was observed in HepG2 cells in either the presence or the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance in negative for cytogenicity under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

There are three in vivo micronucleus assays available with conflicting results. Therefore, in accordance with ECHA Decision number TPE-D-2114428335-52-01/F, an in vivo alkaline comet assay was carried out to clarify the potential for mutagenicity to somatic cells. A statistically significant mild to moderate increase in tail intensity was observed in all tissues at all concentrations tested.

A transgenic rodent assay (OECD TG 488) is proposed to evaluate the potential for mutagenicity to germ cells.

Link to relevant study records
Reference
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
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
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
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
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
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

The available information for the registration substance on in vitro and in vivo genetic toxicity has been considered, and there is evidence for mutagenicity from in vitro studies and for damage to DNA from an in vivo mammalian alkaline comet assay, where a statistically significant mild to moderate increase in tail intensity was observed in all tissues at all concentrations tested. The potential for germ cell mutagenicity has been considered on the basis of all available data for the registration substance and related substances, including: toxicokinetic modelling for the registration substance and measured toxicokinetic data (intraperitoneal administration) for another alkoxysilane; repeated dose toxicity studies for the registered substance; genetic toxicity data with the registered substance and with other silanes and siloxanes with an epoxy group. No clear conclusions about germ cell mutagenicity have been reached, therefore a transgenic rodent assay (OECD TG 488) is proposed. The consideration of all available evidence is discussed in detail below.

Summary of available in vivo data for registration substance 3-(2,3-epoxypropoxypropyl]trimethoxysilane

Method

Route of administration

Result

Toxicity to bone marrow

Systemic effects

Reference

Micronucleus

Intraperitoneal injection

Positive

Yes Moderate to severe reductions of 22 to 59% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the vehicle control animals

Clinical signs included lethargy and piloerection at 1000 and 2000 mg/kg.

BioReliance, 1999

Micronucleus

Intraperitoneal injection

Negative

Toxicity to bone marrow was observed in female mice at 24 h observation.

All animals showed clinical signs following dosing (1600 mg/kg bw as a suspension in corn oil)

Hüls, 1994

Micronucleus

Oral gavage

Negative

No data

No data

Dow Corning Corporation, 1982a

Sister chromatid exchange

Intraperitoneal injection

Negative

No data; a high degree of bioavailability is expected after ip administration

No data

Dow Corning Corporation, 1982d

Sister chromatid exchange

Inhalation

Negative

No data

No data

Dow Corning Corporation, 1982d

 Comet  Oral  Positive  Not applicable  No  Charles River Laboratories, 2020

1. Toxicokinetics: no toxicokinetic data are available for the substance. There is evidence of systemic exposure following intraperitoneal administration from an in vivo bioavailability study for another trialkoxysilane (see section 7.1 of IUCLID and 5.1 of the CSR), but this does not give an indication of bioavailability after oral administration. In addition, systemic toxicity was observed in two of the three micronucleus assays; no information was available for the third study. Lethargy and piloerection were reported following intraperitoneal exposure in one of the micronucleus studies (BioReliance, 1999) at 1000 and 2000 mg/kg bw. In the more recent of the negative micronucleus assay, severe clinical symptoms were observed following intraperitoneal (ip) application (Hüls 1994). No systemic toxicity was observed following oral administration of the registered substance in repeated dose toxicity testing. Therefore there is no information on bioavailability following oral administration.

2. The positive in vivo micronucleus assay is considered not relevant as intraperitoneal administration it is not a physiologically relevant route for this substance, taking into account expected routes of exposure for the substance. There is no clear explanation for the positive result but it is likely that route of exposure is important.

3. The weight of evidence from all studies (micronucleus and sister chromatid exchange), and the agreement of results from studies using a physiologically relevant route of exposure, is that the substance is not mutagenic.

4. In vivo micronucleus assays are not considered appropriate to investigate the mutagenic effects of substances that are mutagenic to bacterial and mammalian cells. This is implied in the REACH Regulation in the column 2 adaptations for in vitro mammalian cell cytogenicity and mutagenicity testing, and is stated in ECHA Guidance R07a. Appropriate assays are, according to the ECHA Guidance (p 364), the transgenic rodent assay (OECD Test Guideline 488) and the in vivo Comet assay (OECD Test Guideline 489) or, if justified, Unscheduled DNA Synthesis, in vivo (OECD Test Guideline 486). There are no in vitro effects that indicate that a micronucleus assay is the appropriate in vivo assay to conclude on somatic cell mutagenicity.

5. Carcinogenicity: the substance was not considered tumorigenic when applied to the clipped skin of mice (25 µl dose of 25% test material in acetone) three times per week for approximately 78 weeks (BRRC, 1982). The ECHA Guidance (ECHA, 2014, R07a p 337) states: “There is considerable evidence of a positive correlation between the mutagenicity of substances in vivo and their carcinogenicity in long-term studies with animals.” As the substance has been shown not to be carcinogenic, it is unlikely that it is mutagenic.

To summarise, the negative data from the in vitro micronucleus test on the structural analogue and the negative in vivo micronucleus results following a physiologically relevant route of exposure, with additional evidence of bioavailability indicate that there is not sufficient evidence to classify as Mutagen Category 2, and so an in vivo comet assay has been carried out. The comet assay can detect evidence of DNA damage that may lead to both gene mutation and chromosome mutation events and is therefore the ideal test method for the follow-up of the in vitro / in vivo genotoxicity profile of the substance (ECHA guidance on mutagenicity (R.7a, 19 August 2014, p 343) states: “[…] the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations) but will also detect DNA damage that may be effectively repaired or lead to cell death". The comet assay can therefore be considered a conservative estimate of genetic toxicity to somatic cells.

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.

Consideration of the need for further testing for germ cell mutagenicity

The evidence from the comet assay of damage to DNA in the liver shows that the test substance reaches the liver cells indicating that the registered substance has the potential for interaction with DNA. However, this is not conclusive evidence that the substance would induce heritable transformation in germ cells, so a transgenic rodent assay (OECD TG 488) is proposed to evaluate the potential for mutagenicity to germ cells.

Strategy for identification of key studies

Where there was more than one result for an endpoint the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. If the results were not in agreement, studies giving positive results were chosen, unless the weight of evidence indicated the result was not indicative of potential for mutagenicity. The results of all the bacterial studies were in agreement and showed evidence of mutagenicity. In mammalian mutagenicity studies, positive results were obtained when tested in L5178Y mouse lymphoma cells (Litton Bionetics, 1983), but negative results when Chinese hamster ovary cells were used (similar to OECD 476, Reliability 4), (Allied Corporation, 1979).

No data are available for the registered substance for in vitro cytogenicity, however, data are available for the structural analogue [3-(2,3-epoxypropoxy)propyl]triethoxysilane (CAS 2602-34-8) from an in vitro micronucleus assay. [3-(2,3-epoxypropoxy)propyl]triethoxysilane has been tested for ability to induce formation of micronuclei in a study conducted according to OECD 487 (Lionti et al., 2014). No evidence of test-substance induced micronucleus formation was observed in HepG2 cells in either the presence or the absence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance in negative for cytogenicity under the conditions of the test. This study is not considered to be key, as an in vivo mammalian alkaline comet assay is available.



Justification for classification or non-classification

There is insufficient evidence to make a conclusion on mutagenicity to germ cells, therefore, a further study is proposed.