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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:
21/02/2019 to 16/05/2019
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Boronic acid, B-(2-fluoro-3-methoxyphenyl)-
EC Number:
609-099-0
Cas Number:
352303-67-4
Molecular formula:
FC6H3(OCH3)B(OH)2
IUPAC Name:
Boronic acid, B-(2-fluoro-3-methoxyphenyl)-
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Off white Powder
CAS No. 352303-67-4
Water Solubility: 3.62 g/L
Batch No. 18362500

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Age at time of initial administration: 6-8 weeks old
Body Weight: Females 166.1-186.7g; Males 175.8-201.6
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Quarantine and Acclimatization:
The animals introduced were quarantined and domesticated for 4 days. During quarantine, the animals were observed for water and food intake, healthy status, as well as for signs of illness and death. After the health status was confirmed by the Attending Veterinarian, animals were assigned to different experimental groups.

Housing Conditions:
Animals were housed in the polypropylene plastic boxes with the cage size of 423 mm x 269 mm. Two animals of the same gender were group-housed in one cage. Room temperature and relative humidity were maintained in the ranges of 20.04 - 23.75°C and 42.91-74.19 %RH, respectively. Lighting was controlled to provide 12 h of light and 12 h of darkness in a 24-hour period. Ventiliation controlled at a rate of at least 15 times of air exchanges per hour. Bedding and cages were replaced twice per week. Diets and drinking water were given to animals ad libitum, unless specified, throughout the course of study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn Oil
Details on exposure:
Five experimental groups were designed in the study with 40 animals (25 males and 15 females) in total, as illustrated in Table below. Group size was 10 animals/ group (5/group/sex) for 3 dosage groups and 5 males/group for 2 control groups. FMPBA was given to animals at three dose levels of 360 mg/kg (high dose), 180 mg/kg (medium dose) or 90 mg/kg (low dose). The positive control was EMS at the dose of 200 mg/kg. The negative control is the formulation vehicle of corn oil.
Duration of treatment / exposure:
Test substance or vehicle was given to animals at Oh, 24h, and 45h for 3 treatments
Frequency of treatment:
Test substance or vehicle was given to animals at Oh, 24h, and 45h for 3 treatments by gastric gavage using a round-tip gavage needle (size 12) and the sterile syringes. Positive control of EMS was given to animals for two treatments at 24h and 45h. The dosage volume was 10 mL/kg. Animals were deprived of food overnight (about 16 hours) before administration, and were restored 1-2 hours after administration. Animals had free access to drinking
water all through the study.

Post exposure period:
Cage-side observations: All animals before grouping and experimental animals after grouping were observed once daily for morbidity and mortality, appearance, fur, activity, reaction, breathing state, posture. Body weight: Animal body weight was weighed using the electronic balance. All animals were weighted upon arrival, group assignment, and before the initial dosing. Tissues were harvested 3 hours after the last dosing. all animals were anesthetized with 100% carbon dioxide (C02) via inhalation.

Doses / concentrationsopen allclose all
Dose / conc.:
90 mg/kg bw/day
Dose / conc.:
180 mg/kg bw/day
Dose / conc.:
360 mg/kg bw/day
No. of animals per sex per dose:
5 male/5 female per dose
5 males for negative and positive control groups
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl Methanesulfonate (EMS)

Examinations

Tissues and cell types examined:
Liver tissue collection: Tissues were removed, dissected and a portion about 5mm3 in the middle of left lobe was collected and weighed immediately for single cell suspension preparation.
Details of tissue and slide preparation:
Single cell suspension preparation: Tissues collected were minced immediately, rinsed with mincing buffer (20 mmol/L EDTA-Na2 in PBS), followed by gently grinded, and sieved through a 70 µm mesh screen. Tissues were maintained on ice during the procedure. Tissue homogenate were then centrifuged (4 'C, 1500 rpm, 10 min) and re-suspended with mincing buffer to make single cell suspensions. Slides precreation: Low melting point agarose (LM) was preheated before use according to the kit manual. Taken 50 µL single cell suspension solutions and added into 500 µL melted agarose, followed by gently mixed with a pipette. Taken 50 µL/well mixture and placed onto slides provide by the kit. One slide labeled individually was prepared for each animal.
Cell lysis: Once prepared, slides were placed into the refrigerator (2-8'C) for 30 minutes under subdued lighting conditions, and immersed in chilled lysing solutions (lysis buffer: DMSO=lO:l) overnight in the refrigerator.
Unwinding and electrophoresis: After incubation, slides were rinsed by purified water and placed into chilled alkaline lysis buffer (pH> 13) for unwinding at room temperature for 20 minutes. Slides were then subjected to electrophoresis under the conditions of 0.7V/cm for 20 minutes in the dark with the pre-cooled electrophoresis buffer (300 mM NaOH, lmM EDTA). After electrophoresis, slides were rinsed by purified water for twice, and dried with absolute ethanol at 37'C for I 5 minutes.
Slides were stained with 100 µL diluted Gene Red staining solutions for staining 30 minutes in the dark. After being dried at 37'C for 15 minutes, slides were examined with fluoresce microscope (200x) and captured with the camera.


Statistics:

The mean and standard deviation of % Tail DNA and hedgehog cells in each group were calculated and plotted using Microsoft Office. The data were expressed as mean ± standard deviation (X ± SD).
Due to uneven variance, the Welch's t-test were used to analyze inter-group difference between negative control (vehicle) group and each dose group or positive control group by SPSS software (IBM, version 19.0). When the p value is less than 0.05 (p<0.05), the inter-group difference is considered statistically significant, and p<0.01 as highly statistically significant.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
In animals given with FMPBA at dose levels of 90 mg/kg, 180 mg/kg, and 360 mg/kg, the% tail DNA was 1.28±0.56, 1.17±0.21, and 1.77±0.49
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
One female animal (#39) in the medium dose group was found dead after the 2nd administration of test substance. Necropsy was performed and the causes of death was found to be due to gavage dosing procedures and thus determined not related to the treatment. No other animals were found morbid or moribund during the study.

Throughout the course of the study, no clinical signs were observed to be related with test substance administration. Individual cage-side observations results were presented

Applicant's summary and conclusion

Conclusions:
Under the testing conditions of this study, the test substance FMPBA within the dose range of 90-360 mg/kg b.w. did not induce DNA strand breakage in the liver of rats.
Executive summary:

This study was designed according to theTesting Guidance of 489"Invivo Mammalian Comet Assay, OECD Guideline for the Testing of Chemicals, 2014".The objective  of the study  was to measure DNA strand breaks  in  rats orally administrated with test substance of FMPBA and evaluate its potential genotoxic risks.

Sprague Dawley (SD) rats were used in the study and randomized into five treatment groups:three dosage groups with 10 animals/group (5/group/sex) and two control groups, i.e. negative control and positive control groups, with 5  male animals/group. The test substance FMPBA was given to animals at the dose of 360mg/kgb.w., 180 mg/kgb.w.or 90mg/kgb.w. by gastric gavage at Oh24h and 45h for 3 times.Animals in negative control group were given with vehicle of corn oil in the same manner as test substance.  Animals positive control group were given twice with 200mg/kg ethyl methanesulfonate (EMS) at 24h and 45h. Liver tissues were collected from all animals within 3 hours after the last administration. Single cell suspension was prepared on slides for staining and microscopic examination. For each sample, 150 cells excluding hedgehogs were analyzed on the % tail DNA ,tail length and tail moment. The % tail DNA was used as indicator to evaluate DNA strand breaks caused by the test substance.

Results showed tha tthe % tail DNA in positive group was 17.55±4.04, which was significantly  higher than that of negative control group of corn oil 0.67 +/- 0.13 (p<0.01), indicating that the test system was reliable in this study. In animals given the given with FMPBA,  the % tail DNA was 1.28±0.56,  1.17±0.21,  and 1.77±0.49 for the dose levels of 90mg/kg, 180 mg/kg, and 360 mg/kg, respectively, all of which were not significant different from that of negative control group (p>0.05) and there was no dose-related relevance.The test chemical is thus considered negative

in this comet assay.