2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-

Genetic toxicity: in vivo

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:

28 November 2017 to 06 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

3-trimethoxysilylpropyl methacrylate

EC Number:

219-785-8

EC Name:

3-trimethoxysilylpropyl methacrylate

Cas Number:

2530-85-0

Molecular formula:

C10H20O5Si

IUPAC Name:

3-(Trimethoxysilyl)propyl 2-methylprop-2-enoate

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was disolved in deionized water/acetic acid 50/50 mixture.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The hydrolysed test substance formulation used for generation of exposure atmospheres was prepared for dosing as a 15% mixture in stock water solution by adding the test substance to the stock water and stirring until clear. Once clear, the pH was checked to ensure that the final pH was between 3.0 to 3.3. The hydrolysed test substance formulation was prepared on each day of use. No analysis of test solution was carried out, however, the hydrolysate preparation would have contained some parent substance.
- Preliminary purification step: none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 200-320 grams (males) / 150-250 grams (females)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material in an environmentally controlled room.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was offered ad libitum during the study, except during exposure periods, acclimation to nose-only restraint tubes, and designated periods of fasting.
- Water: Reverse osmosis-treated water was available, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10 / hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water (acetic acid), pH 3. The vehicle, deionized water (pH 3 ± 0.1) and also referred to as stock water solution, was prepared twice using glassware cleaned according to Sponsor-provided instructions and stored at room temperature (18°C to 24°C). Details of the preparation and dispensing of the vehicle for the test substance have been retained in the Study Records.
JUSTIFICATION: not specified
In the substance evaluation final decision, it is stated that “ECHA considers that the in vivo comet assay should be conducted with an aerosolised atmosphere of the registered substance which mimics the worst case test conditions of the aerosol inhalation repeated dose toxicity studies reported in the registration data.” The worst-case test conditions are the highest achievable concentration at the lowest pH used in the repeated dose studies, with the test substance stirred for one hour, therefore the vehicle was chosen to produce a hydrolysate of the test substance at pH 3.

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only
Test substance preparation and animal exposure tool place in Charles River Test Facility

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a stainless steel, conventional nose-only exposure system
- Method of holding animals in test chamber: Animals were kept in nose-only restrained tubes.
- Source and rate of air: Exposure system air was supplied from a breathing quality in-house compressed air source and/or a HEPA- and charcoal-filtered air source. Airflow rate through the exposure system was set based on output from the aerosol generator and the dilution airflow, and provided a sufficient volume for the number of animals to be exposed and for exposure atmosphere sampling. The airflow rates for the nose-only system were calculated from calibration curves for the generation device and/or flowmeters.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: not specified
- Temperature, humidity, pressure in air chamber: The target range for average temperature and relative humidity of the exposure atmosphere were 22 ± 3ºC and 50 ± 20%, respectively.
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: Aerosol particle size determinations were conducted at least twice utilizing a cascade impactor. Pre-weighed collection substrates were loaded into the impactor prior to sample collection. Following sample collection, the substrates were re-weighed and the particle size was calculated based on the impactor stage cut-offs. The aerosol particle size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1 to 3.0 µm and GSD is 1.5 to 3.0
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: Following sample collection, the sample was re-weighed and the wet weight was recorded. The wet weight was only used to guide the exposures, not to define exposure concentrations. Following wet weight determination, the filter sample was first dried by placing it in an oven set to approximately 120 °C for 20 minutes and then cooled for 10 minutes in a desiccator. Following the drying and cooling period, the filter was re-weighed. The aerosol exposure concentration (mg/m3) was calculated by dividing the gravimetrically determined mass of aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final dried weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. The adjusted exposure concentrations (equivalent MPTMS concentration) were calculated by dividing the aerosol exposure concentration by 0.7217. This correction factor represents the ratio of silicone formed on the dried filter relative to the MPTMS.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours a day

Frequency of treatment:

once a day for two consecutive days

Post exposure period:

between 2 and 4 hours following last exposure

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/m³ air

Doses / concentrations 2

Dose / conc.:

500 mg/m³ air

Doses / concentrations 3

Dose / conc.:

1 000 mg/m³ air

No. of animals per sex per dose:

6 males and 6 females per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate
- Justification for choice of positive control(s): A known substance that induces DNA strand breaks.
- Route of administration: oral (gavage)
- Doses / concentrations: The ethyl methanesulfonate formulation was prepared for dosing as a weight-to-volume mixture in 0.9% saline at a concentration of 200 mg/kg (10 mL/kg volume).

Examinations

Tissues and cell types examined:

Immediately following euthanasia, the five (5) surviving rats in Groups 1-5 had nasal tissue, lung and liver collected and examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The target exposure concentrations were selected by the Sponsor Representative in consultation with the Study Director based, in part, on the a previous acute inhalation toxicity study in rats. In that previous study, hydrolysed MPTMS aerosol was administered as a single 4-hour exposure at an achieved concentration of 2280 mg/m3. No deaths were observed.
In order to assess the potential for exposure-limiting toxicity, a range-finding study was carried out using a high target adjusted aerosol concentration (equivalent MPTMS concentration) of 1450 mg/m3 was selected in an effort to define the maximum tolerated dose (MTD). During method development, an actual maximal feasible concentration was determined and the target concentration for the high-dose was updated or confirmed. The mid and low target aerosol concentrations were selected to be one-half (1/2) and one-quarter (1/4) of the selected high-dose, respectively. The concentrations achieved in the dose range-finder were 0, 256, 523, and 1043 mg/m3.
All animals survived until the scheduled euthanasia (~2 hours post 2nd exposure).
Following each exposure, there were observations related to respiration in the high-dose group: labored respiration, increased respiration, gasping, and/or rales. These findings were not considered to be dose limiting.
The exposure levels for the definitive phase were selected based on data from the dose range finding phase (mortality/moribundity, body weights, etc.). The high exposure concentration was the highest achievable non-lethal exposure concentration (maximum tolerated dose) based on the results of the dose range-finding phase. The mid and low target aerosol concentrations were selected to be one-half (1/2) and one-quarter (1/4) of the selected high dose, respectively.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treatment and euthanasia of animals was carried out at Charles River Test Facility by Charles River technicians, who removed head, liver and lungs. Samples of the nasal tissue, right lung and liver were collected by BioReliance staff at the Charles River Test Facility.

DETAILS OF SLIDE PREPARATION: A section of each of the tissues were placed in 3 mL of chilled mincing solution, then minced with fine scissors to release the cells. The cell suspensions were strained into a pre-labelled conical polypropylene tube through a Cell Strainer and were kept on wet ice during preparation of the slides.
From each liver, lung and nasal tissue cell suspension, an aliquot of 2.5 µL was mixed with 75 µL (0.5%) of low melting agarose. The cell/agarose suspension was applied to microscope slides. Commercially purchased pre-treated, multi-well slides were used and these slides have 20 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Sliders were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two Trevigen, Inc 20-well slides were prepared per animal per tissue. Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.
Slides of the processed nasal tissue, lung, and liver prepared by BioReliance personnel at the Charles River Test Facility were stored at room temperature with desiccant. Slides were shipped on the first available Monday, Tuesday, or Wednesday at ambient temperature to BioReliance by overnight shipment.

METHOD OF ANALYSIS: Analysis of slides was carried out at the BioReliance Test site. Three wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV.
The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100].
Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

Evaluation criteria:

All conclusions were based on sound scientific judgment. As a guide to interpretation of the data, the following were considered:
• The test substance was considered to induce a positive response in a particular tissue if the mean % tail DNA (or other parameters of DNA damage) in one or more test substance groups (doses) was statistically elevated relative to the concurrent negative control group.
• The test substance was judged negative for induction of DNA damage if no statistically significant increase in the mean % DNA damage (or other parameters) in the test substance groups relative to the concurrent negative control group was observed.
However, the results of the statistical analysis may not be the only criterion in determination of the test substance potential to induce DNA damage. The following may be taken in consideration:
• The historical vehicle control data; a statistically significant increase in the mean % DNA (or other parameters) was not considered biologically relevant if the values did not exceed the range of historical vehicle control.
• Because cells undergoing necrosis or degeneration are prone to DNA degradation, independent of direct genotoxic effects of the test substance, doses that were found to be cytotoxic, by histopathology evaluation, were not considered as relevant doses and were not taken in consideration during the generation of the study conclusions. Accordingly, any statistically significant increase in DNA damage occurring at a cytotoxic dose was not considered as a positive finding.
• A dose-dependent increase in the mean % tail DNA (or other parameters) across the dose levels tested; if there was evidence of a dose-response with no evidence of a statistically significant increase, additional testing, including histopathology evaluation of the tissue, was considered.

Statistics:

In order to quantify the test substance-related effects on DNA damage, the following statistical analysis was performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA (or other parameters of DNA damage) generated for the negative control (filtered air control) and test substance-treated groups were compared using Levene’s test (significant level of p ≤ 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test were performed (significant level of p < 0.05).
• Linear regression analysis was used to determine a dose-response relationship (p < 0.01).
• A pair-wise comparison (Student’s T-test p 0.05) was used to compare the data from the positive control group against the negative control group.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
- Dose range: A dose range finding study has been completed using mean concentrations of 0, 256, 523, and 1043 mg/m3.
- Solubility: not specified
- Clinical signs of toxicity in test animals: Following each exposure, there were observations related to respiration in the high-dose group: labored respiration, increased respiration, gasping, and/or rales. While present, these type of findings were not considered to be dose limiting. All animals survived until the scheduled euthanasia.

Any other information on results incl. tables

RESULTS OF DEFINITIVE STUDY

Liver cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was ≤ 1.4%, which was comparable with the % of clouds in the negative control group (0.8%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the liver

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was ≤ 2.4%, which was higher than the % of clouds in the negative control group (1.0%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the liver

Lung cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and mid test substance groups was 0.4%, which was comparable with the % of clouds in the negative control group (0.4%). The high test substance group was 1.6% which was higher than the % clouds in the negative control group.  

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• A statistically significant increase response in the % Tail DNA (DNA damage) was observed in the test substance group (500 mg/m3) relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05); however this increase was not biologically significant and was within the Test Site’s historical control for this tissue, thus it was not considered to be biologically relevant.  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the lung

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was ≤ 3.2%, which was lower than the % of clouds in the negative control group (4.2%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• A statistically significant decrease response in the % Tail DNA (DNA damage) was observed in the test substance group (1000 mg/m3) relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05).  This was not considered to be biologically relevant, since the study was designed to detect and evaluate increase in the DNA damage, which would be evident by an increase in % tail DNA.

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the lung

Nasal cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and high test substance groups was ≤ 12.4 %, which was higher than the % of clouds in the negative control group (8.4%). The mid test substance group was 6.8% which was lower than the % clouds in the negative control group.

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the nasal

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and mid test substance groups was ≤ 34.8%, which was higher than the % of clouds in the negative control group (24.4%). The high test substance group was 17.4% which was lower than the % of clouds in the negative control group.

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal cells as compared to the negative control group (Student’s t test, p ≤ 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the nasal

Applicant's summary and conclusion

Conclusions:

In an in vivo Comet Assay, conducted according to OECD test guideline 489 and in compliance with GLP, 3-trimethoxysilylpropyl methacrylate (aerosolized, hydrolysed) was tested for the ability to induce DNA damage in Sprague-Dawley rats. No test-substance induced DNA-damaging response was observed in nasal tissue, lung and liver from male and female rats following 6-hour nose-only inhalation exposure with the test substance for two consecutive days. None of the test substance-treated animal slides had significant increases in the % Tail DNA compared to the respective negative control. The negative control % Tail DNA was within the laboratory’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the negative control. Thus, all criteria for a valid assay were met for liver, lung and nasal tissue. It was concluded that the test substance was negative for DNA damage in liver, lung and nasal tissue in the in vivo Comet Assay.