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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:
31 Jan 2022 - 28 Apr 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
m-phenylene di(acetate)
EC Number:
203-596-2
EC Name:
m-phenylene di(acetate)
Cas Number:
108-58-7
Molecular formula:
C10H10O4
IUPAC Name:
3-(acetyloxy)phenyl acetate
Specific details on test material used for the study:
Physical Description: Clear pale yellow liquid
Storage Conditions: At room temperature protected from light

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han
Details on species / strain selection:
The Wistar-Han rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity test by regulatory agencies.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-10 weeks
- Body weight: The body weights of the rats at the start of the treatment were within 20% of the sex mean (295.3 - 310.0 g)
- Assigned to test groups randomly: yes
- Fasting period before study: A limited quantity of food was supplied during the night before last dosing (approximately 7 g/rat).
- Housing: Polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together. Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in, pater and/or objects for chewing, except when interrupted by study procedures/activities
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH pellets ad libitum, except during designated procedures
- Water: Municipal tap water, ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

Following analyses, it was considered that there were no known contaminants in feed, water and enrichment that could have interfered with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24°C (target), 21 to 22 °C (actual)
- Humidity: 40 to 70% (target), 45 to 57% (actual)
- Air changes: 10 or more air changes per hour
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 31 Jan 2022 To: 07 April 2022

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
PREPARATION OF THE DOSING SOLUTIONS:
No correction was made for the purity/composition of the test material. An adjustment was made for specific density of the test material (factor of 1.1758).
Depending on the concentration, the test material was dissolved or suspended in corn oil. The specific gravity of corn oil is 0.92 g/mL. Test material concentrations were vortexed, treated with ultra-sonic waves and stirred to obtain a homogeneous suspension or until the test material was completely dissolved.
This resulted in a yellow translucent suspension for the formulations with a concentration of 200 mg/mL and yellow solutions for the formulations with a concentration of 50 and 100 mg/mL
Test material concentrations were dosed within 3.5 hours after preparation. Any residual volumes were discarded.
The first day of dosing was designated as Day 1. The doses were given using a plastic feeding tube. The dosing volume was 10 mL/kg body weight.

DOSE-RANGE FINDING STUDY:
Selection of an adequate dose-range for the Comet main test was based on a dose-range finding study. The test procedure and conditions were similar to those applied in the main test.
In the dose-range finding study, one dose-group was used to define the MTD (Maximum Tolerated Dose) based on the toxic signs observed after dosing with different doses of the test material.
One dose group, comprising of 3 males and 3 females, was dosed for two consecutive days (once daily) with the highest concentration of test material that was used for the main study. The observation period after dosing was one to three days. During this period mortality and physical condition were recorded at least once a day.
Based on the results of the dose-range finding test, there were no substantial differences in toxicity between sexes, and as such only male animals were used in the main study. Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg bw/day were selected as appropriate doses for the main test.


Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
Once daily
Post exposure period:
3-4 hours after the last dose of the treatment with the test material, vehicle (negative control) or EMS (positive control), the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and, the liver, duodenum, and glandular stomach were collected/isolated and examined for DNA damage with the alkaline Comet assay.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
- Main study, group 4 (2000 mg/kg bw/day): 8
- Main study, group 5 (positive control): 3
- Main study, remaining treatment groups: 5
- TK study (proof of exposure): 3
Control animals:
yes, concurrent vehicle
Positive control(s):
ethyl methanesulfonate (EMS, Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline (Group 5).

Examinations

Tissues and cell types examined:
liver, duodenum and glandular stomach
Details of tissue and slide preparation:
ISOLATION OF CELLS

Liver
The isolation method was based on the publication of Hu et al (2002). A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase dissolved in HBSS (Ca2+ - and Mg2+-free) and incubated in a shaking water bath at 37°C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells
The isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca2+, Mg2+ free). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study.
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodenum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded.
The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca2+, Mg2+ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use).
The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

PREPARATION OF SLIDES
To the cell suspension, melted low melting point agarose was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15 to 32 minutes in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.


LYSIS, ELECTROPHORESIS AND STAINING OF THE SLIDES
The cells on the slides were overnight (approximately 17-18h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator (at 4°C). After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volts/cm. The electrophoresis was performed for 20 (stomach, duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolute ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

SAMPLING, FIXATION AND STORAGE OF TISSUE FOR HISTOTECHNOLOGY AND HISTOPATOLOGY
Part of the liver, glandular stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was ultimately required.

COMET SCORING
To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code was placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
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.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control.


ACCEPTABILITY CRITERIA
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are 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.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg bw/day

Evaluation criteria:
A test material 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 is detected compared with the concurrent negative control.
b) There is dose-related increase when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test material is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Statistics:
ToxRat Professional v 3.3.0 was used for statistical analysis of the comet assay data.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals.
Toxicity:
yes
Remarks:
Clinical observations were made in the groups treated with 1000 and 2000 mg / kg bw (tremors and lethargy). Three animals in the groups treated with 2000 mg / kg bw died on day 1 postdose.
Vehicle controls validity:
valid
Remarks:
The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats within the 95% control limits of the distribution of the historical control data for the vehicle control
Positive controls validity:
valid
Remarks:
The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database
Additional information on results:
DOSE-RANGE FINDING STUDY
In the dose-range finding test, male and female animals dosed with 2000 mg test material per kilogram body weight showed treatment related clinical signs (convulsions, pinched eyes, head shaking and shivering).
The Maximum Tolerated Dose (MTD; described as the dose that will not kill the animals but will provoke signs of toxicity) was confirmed to be 2000 mg/kg bw.

MORTALITY AND TOXIC SIGNS
The animals of the groups treated with 500 mg /kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Clinical observations were made in the groups treated with 1000 and 2000 mg / kg bw (tremors and lethargy). Three animals in the groups treated with 2000 mg test material/ kg bodyweight died on day 1 postdose.

COMET SLIDE ANALYSIS
Comet slides were prepared and analyzed. No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. In addition there were no Hedgehogs observed in vehicle and test material treated groups. The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.86 ± 0.90% (mean ± SD), 4.20 ± 0.45% (mean ± SD) and 6.97 ± 2.15% (mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control (Table 25). The positive control (EMS) induced a significant increase and showed a mean Tail Intensity of 65.27 ± 2.96 (mean ± SD, p<0.001 Students t test), 35.24 ± 5.33% (mean ± SD, p<0.001 Students t test) and 51.19 ± 2.13% (mean ± SD; p<0.001 Students t test) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

BIOANALYSIS
Blood was sampled 1, 2, 4, 6 and 24 h after the second dose of satellite animals dosed with the vehicle and the highest concentration of the test material. Substance A was analyzed as indirect proof of exposure for the test substance, since the test substance was unstable in plasma. Exposure was demonstrated in plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples.

Any other information on results incl. tables

Dose Formulation Analysis


Accuracy


The concentrations analyzed in the formulations were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). No test material was detected in the vehicle.


Homogeneity


The formulations of the lowest and highest dose group were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Overview Tail Intensity in Liver Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



2.86



0.90



Test Material 500 mg/kg bw



2.74



0.80



Test Material 1000 mg/kg bw



2.21



0.32



Test Material 2000 mg/kg bw



2.06



0.41



EMS 200 mg/kg bw



65.27



2.96



 


Overview Tail Intensity in Duodenum Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



4.20



0.45



Test Material 500 mg/kg bw



3.70



0.39



Test Material 1000 mg/kg bw



3.86



0.90



Test Material 2000 mg/kg bw



5.42



1.60



EMS 200 mg/kg bw



35.24



5.33



       
Overview Tail Intensity in Glandular Stomach Cells of Male Rats



































 

Tail Intensity (%)



S.D.



Vehicle Control



6.97



2.15



Test Material 500 mg/kg bw



5.23



1.76



Test Material 1000 mg/kg bw



5.31



2.06



Test Material 2000 mg/kg bw



4.49



1.88



EMS 200 mg/kg bw



51.19



2.13



 


Historical Negative Control Data for Comet Assay










































 



Duodenum
Tail Intensity (%)


Males and Females



Liver
Tail Intensity (%)


Males and Females



Glandular Stomach
Tail Intensity (%)


Males and Females



Mean



6.7



2.5



5.9



SD



3.2



1.4



3.4



n



26



41



30



Lower control limit


(95% control limits)



0.4



-0.1



-0.7



Upper control limit


(95% control limits)



13.1



5.2



12.5



SD = Standard deviation


n = Number of observations


 


Historical control data from experiments performed in November 2018 - November 2021


 
Historical Positive Control Data for Comet Assay










































 



Duodenum
Tail Intensity (%)


Males and Females



Liver
Tail Intensity (%)


Males and Females



Glandular Stomach
Tail Intensity (%)


Males and Females



Mean



51.1



84.2



57.9



SD



10.0



6.3



9.7



n



26



42



30



Lower control limit


(95% control limits)



31.4



71.9



38.9



Upper control limit


(95% control limits)



70.7



96.5



76.8



SD = Standard deviation


n = Number of observations


 


Historical control data from experiments performed in November 2018 - November 2021


 


 

Applicant's summary and conclusion

Conclusions:
The comet assay is valid and the test substance is not genotoxic in the Comet assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing of male rats, exposed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw/day (the maximum tolerated dose).
Executive summary:

A comet assay was performed in male rats according to OECD guideline 489 and in accordance with GLP principles. 


 


The objective of this study was to obtain information on the potential genotoxicity of  the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.


 


Study design


The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.


 


The test material was a clear pale yellow liquid. The test material was dissolved or suspended in corn oil, depending on the concentration.


 


No test material was detected in the vehicle. The concentrations analyzed in the formulations were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration). The formulations of the lowest and highest dose group were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg bw/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes in a dose range finding study only males were used in the main study.


 


In the main study male animals were dosed with vehicle (corn oil), test material (at 500, 1000 and 2000 mg/kg body weight/day) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight.


 


Clinical signs of toxicity were limited to the middle and high dose group and included tremors and lethargy.


 


In addition, blood for bioanalysis of the test substance in plasma was collected from satellite animals for the 2000 mg/kg bw/day group (highest dose group) and from satellite animals for the vehicle control group. Substance A was analyzed as indirect proof of exposure for the test substance, since the test substance was unstable in plasma.


 


Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.


 


Results


No statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals.


 


The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 2.86 ± 0.90% (mean ± SD), 4.20 ± 0.45% (mean ± SD) and 6.97 ± 2.15%
(mean ± SD) in male animals, respectively, which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 65.27 ± 2.96%
(mean ± SD), 35.24 ± 5.33% (mean ± SD) and 51.19 ± 2.13% (mean ± SD) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Exposure was demonstrated in plasma samples from all test material-dosed animals after dose administration. No measurable amount of analyte was detected in control rat plasma samples. Hence, all criteria for an acceptable assay were met.


 


Conclusion


In conclusion, the test is valid and the test material is not genotoxic in the Comet assay, as shown in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions used in this study.