3-Fluorphenylboric acid

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:

2016-03-24 to 2016-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

09-2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

09-2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

4-Ethoxy-2,3-difluorphenylboronic acid

EC Number:

606-729-6

Cas Number:

212386-71-5

Molecular formula:

C8 H9 B F2 O3

IUPAC Name:

4-Ethoxy-2,3-difluorphenylboronic acid

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Raleigh, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 213 - 244 g
- Housing: single-housed in high polysulfone cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 17 - 47
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From day 1 - 3

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Methocel® K4M Premium

Details on exposure:

Vehicle Control: The vehicle control article was 0.25% Methocel® K4M Premium Hydroxypropyl Methylcellulose. The stability of the test item in the vehicle has been demonstrated at least 24 hours at room temperature. Therefore, the test article dose formulations were prepared freshly on each day of dosing. Samples of the dose formulations and the vehicle control that were used for the comet/MN assay were collected for analysis.
Dose Formulation Analysis: Test article dose formulations were accurate since the measured concentrations of the test item were within 15% of their nominal values, which was within the predefined acceptance limits (+/- 15% of nominal). No test article was detected in the vehicle control samples. The quantification of the test item was performed using a HPLC method with UV detection.
Dose Administration: The dose volume for each dose administration was 10 mL/kg.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

once daily

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5 m

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group received a single intraperitoneal injection of cyclophosphamide (CP) 24 hours before necropsy. The same group of animals also received a single oral administration of ethyl methanesulfonate (EMS) 4 hours before necropsy. The dose volume for each administration was 10 mL/kg.
Comet Assay: EMS was prepared fresh in dH2O on the day of dosing and administered at a concentration of 300 mg/kg BW.
Micronucleus Assay: CP was prepared fresh in dH2O on the day of dosing and administered at a concentration of 25 mg/kg BW.

Examinations

Tissues and cell types examined:

Evaluated samples
Comet assay: liver, glandular stomach and duodenum
Micronucleus assay: bone marrow
Histology: liver, glandular stomach and duodenum

Details of tissue and slide preparation:

Comet Assay
For each sample, four comet slides (replicates B-E) were prepared and after at least one hour in lysis, liver, duodenum and glandular stomach slide replicates B-E were electrophoresed under alkaline (pH >13) conditions. Prior to electrophoresis, slides were rinsed with 0.4M Tris (pH 7.5) and submerged in alkaline electrophoresis buffer for 20 minutes at 4 - 4.6°C to unwind the DNA. After unwinding, slides were electrophoresed at 4.1 - 5.9°C for 40 minutes at a constant voltage of 0.7V/cm. At the start of electrophoresis, the buffer level was adjusted as necessary to achieve a starting current of approximately 300 mA. After electrophoresis, the electrophoresed slides were neutralized with 0.4M Tris buffer (pH 7.5), dipped in ethanol, air dried and stored at room temperature.
Slides were stained with SYBR Gold™ stain and unless precluded by poor cell density and/or poor sample/slide quality, 150 cells per sample (75 cells per slide, if possible) were scored using the Komet© Image Analysis System. The image analysis version and settings were documented in the raw data. For each sample, 150-182 cells were visually scored for the frequency of ghosts defined as cells with a heavily diffused tail and a non-existent head. Slides were scored without knowledge of the sample treatment group. Any extra replicate comet slides remaining in lysing after comet analysis is complete were not retained. After analysis of the comet slides is complete, the continued quality of the analyzed slides cannot be ensured after >1 month of storage. Therefore, the comet slides were not retained or archived after the report was finalized.

Low Molecular Weight (LMW) DNA Diffusion Assay
To assess for LMW DNA diffusion (%LMW), 1 slide (Replicate A) per comet sample was prepared and lysed for the LMW diffusion assay. After 1-2 hours in lysis, the LMW slides were neutralized with 0.4M Tris buffer (pH 7.5), dipped in ethanol and air dried. Air dried slides were stored at room temperature. After staining slides with SYBR Gold™ stain, 100 cells per sample were scored visually for diffusion and classified using the following categories: I = condensed DNA; II = diffused DNA. Slides were scored without knowledge of the sample treatment group. Since the positive control EMS can be both genotoxic and cytotoxic at the dose it is administered, the %LMW data for the EMS exposure is not considered relevant to the validity of a positive response detected by comet.

Micronucleus Assay
To determine the number of MN-PCEs indicative of clastogenicity or aneugenicity and to assess for the presence of bone marrow suppression indicative of cytotoxicity, at least two MN slides from each animal were analyzed. After staining slides with acridine orange (and unless precluded by low cell density and/or excessive debris), 4000 PCEs per animal were scored for the incidence of micronuclei and 1000 erythrocytes per animal were scored for the percentage of PCEs. Slides were scored without knowledge of the sample treatment group.

Histopathology
Histopathology liver, duodenum and glandular stomach samples from dosed rats and the vehicle control group were analyzed for the presence of cytotoxicity. Tissue samples that had been immersed in fixative were transferred to a different test site for processing and analysis. Tissue samples were processed, embedded in paraffin blocks, sectioned and stained with hematoxylin and eosin. Samples were analyzed by microscopic evaluation for minimal to moderate inflammation and signs of tissue damage or degradation by a board certified veterinary pathologist.

Evaluation criteria:

Criteria for a Valid Test
-For negative studies, the concurrent positive control must induce a statistically significant (p<0.05) increase in DNA damage as determined by the %Tail or MN-PCEs when compared to the concurrent vehicle control.
-The concurrent negative control must be considered acceptable for addition to the Helix3 historical control database by providing a sufficient dynamic range to detect a positive effect.

Criteria for a Positive Response
The test article may be classified as positive for inducing genotoxicity if the following criteria are met:
a.) a statistically significant increase (indicative of strand breaks) or decrease (indicative of crosslinks) in DNA migration and/or an increase in the incidence of micronuclei is detected at one or more dose concentrations and
b.) a statistically significant dose dependent response is detected in the same endpoint and sample type
Regardless of the parameter (e.g. %Tail vs. OTM), comet data collected by image analysis is measured on a continuous scale with an infinite and continuously expanding range of historical values. Therefore, only concurrent control groups within the same experiment were compared to test article dose groups for the determination of a positive response.
A test article may be classified as equivocal for inducing genotoxicity if either criteria (a) or (b) are met, but not both. If an increase in cytotoxicity is detected in the same tissue and dose concentration(s) at which DNA damage is significantly affected, cytotoxicity may be considered a confounding factor in the determination of genotoxicity and a repeat test with the same and/or lower doses may be recommended to verify the presence of genotoxicity in the absence of cytotoxicity. If neither criteria (a) nor (b) are met and direct or indirect evidence of target organ exposure can be demonstrated, the test article may be classified as negative for inducing genotoxicity.

Statistics:

Standard statistical methods incl. Analyse-It software were used

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Comet assay: Negative for inducing genotoxicity in the liver and glandular stomach. The duodenum was assessed as an inappropriate tissue to test with this experimental design.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

ambiguous

Remarks:

Micronucleus assay: Equivocal in the bone marrow of treated animals when dosed up to the toxic dose.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Comet/MN Assay
Minor clinical observations (e.g. lethargy, irregular breathing, partially closed eyelids, porphyrin staining, tremors and spasms) were noted in two animals in the 1000 mg/kg dose group. During necropsy, bloating and distension in the stomach and duodenum was noted in one these two rats. Compared to the vehicle control dose group, there was a 25, 5, 42 and 164% decrease in average body weight gain in the 30, 100, 300 and 1000 mg/kg test article dose groups, respectively.
The liver (%Tail only), duodenum, glandular stomach and bone marrow %MN-PCE data were normally distributed. The liver %LMW and bone marrow %PCE data were not normally distributed. The toxic dose was determined to be 1000 mg/kg based on liver mucosal degeneration in histopathology samples and the 164% decrease in average body weight gain. To determine the presence of a linear dose response in the absence of the toxic dose group, additional trend tests were performed excluding the 1000 mg/kg dose group for all tissues.

Liver Comet
Based on statistical analysis using pairwise comparisons, neither a significant increase nor decrease in DNA migration or %LMW was detected in the liver at any dose concentration evaluated. Based on trend tests, a dose-dependent increase was detected in DNA migration, but no response (increase or decrease) was detected in %LMW. When the toxic dose group was excluded from statistical analysis, no dose-dependent response was detected in DNA migration or %LMW. EMS induced a significant increase in DNA migration and %LMW.

Duodenum Comet
Based on statistical analysis using pairwise comparisons, a significant decrease in DNA migration was detected in the duodenum at 30 and 1000 mg/kg dose concentrations. A significant decrease in %LMW was detected at 100 and 1000 mg/kg dose concentrations. Based on trend tests, no dose-dependent response was detected in either endpoint with or without the toxic dose group included in analysis. EMS did not induce a significant increase in DNA migration compared to the vehicle control. This was due to a higher than average background level of DNA migration in the vehicle control group. Since higher background levels of DNA migration in the duodenum have been associated with multiple administrations of viscous vehicles such as Methocel K4M Premium, the duodenum was an inappropriate tissue for this study design resulting in an invalid test for this tissue.

Glandular Stomach Comet
Based on statistical analysis using pairwise comparisons, no response in DNA migration or %LMW was detected in the glandular stomach at any dose concentration evaluated. Based on trend tests, no dose-dependent response was detected in either endpoint with or without the toxic dose group included in analysis. EMS induced a significant increase in DNA migration but neither an increase nor decrease in %LMW.

Bone Marrow MN
Based on statistical analysis using pairwise comparisons and trend tests, a significant and dose-dependent increase in MN-PCEs was detected at 30 and 1000 mg/kg dose concentrations. A significant dose-dependent decrease in %PCEs was not detected at any dose concentration. When the toxic dose group was excluded from statistical analysis, no dose-dependent response was detected in either endpoint. CP induced a significant increase in MN-PCEs without a decrease in %PCEs.

Histopathology Evaluation
Degeneration of liver hepatocytes was present in two males in the 1000 mg/kg dose group and considered related to oral administration of the test item. Degeneration of hepatocytes consisted of a spectrum of changes including homogenization of cytoplasm, absence of cytoplasmic detail, nuclear fragmentation, and retention of cytoplasmic membranes. A single gross finding was reported in one of these two rats and included distension and bloating of the stomach and duodenum. Examination of the stomach and duodenum did not reveal any abnormalities and no microscopic correlates were present for the gross findings recorded.

Any other information on results incl. tables

Objective

This study was intended to evaluate the potential of the test item to induce genotoxicity in the liver, duodenum, glandular stomach and bone marrow of male Sprague Dawley rats following oral administration. Among the various mechanistic-based techniques for evaluating the genotoxic activity of a test article, the comet assay can detect DNA damage (strand breaks, alkali-labile sites, cross-linking, adduct formation) in individual cells from any target organ and regardless of mitotic activity. Combined with or supplemental to the in vivo micronucleus (MN) assay which can detect clastogens and aneugens in the bone marrow and peripheral blood, this study provided the best method for determining the genotoxicity of the test article.

Study design

Groups of 5 male Sprague Dawley rats were dosed orally once daily via gavage with 0, 30, 100, 300 or 1000 mg/kg over 3 consecutive days. The sample time was 4 hours after last dosing and the evaluated samples in the comet assay were liver, glandular stomach, duodenum and in the micronucleus assay the bone marrow. A histopathological examination in liver, glandular stomach and duodenum was performed.

Results

In the comet assay, there was no response in the liver or glandular stomach. In the MN assay, a significant and dose dependent increase in MN-PCEs was induced in the bone marrow at 1000 mg/kg. However, since liver toxicity and a significant decrease in body weight gain was also detected at this dose level, the increase in micronuclei may be due to toxicity. With the exclusion of the toxic dose from statistical analysis, there was no dose-dependent increase in MN-PCEs. A significant increase in MN-PCEs at a dose level of 30 mg/kg was within the historical control range for the vehicle control and is most likely a statistical artefact.

Conclusion

Under the experimental conditions of this study, the test item was negative for inducing genotoxicity in the liver and glandular stomach and equivocal in the bone marrow of treated animals when dosed up to the toxic dose. Since the duodenum was an inappropriate tissue to test with this experimental design and a valid test was achieved in the glandular stomach as the site of contact, a repeat test in the duodenum is not recommended.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test item was negative for inducing genotoxicity in the liver and glandular stomach and equivocal in the bone marrow of treated animals when dosed up to the toxic dose. Since the duodenum was an inappropriate tissue to test with this experimental design and a valid test was achieved in the glandular stomach as the site of contact, a repeat test in the duodenum is not recommended.