4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02/2022 - 09/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 474.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

EC Number:

929-209-3

Cas Number:

1123312-95-7

Molecular formula:

C14 H13 B F2 O2

IUPAC Name:

4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 474 “Mammalian Erythrocyte Micronucleus Test“, adopted July 29, 2016, recommends using mice. The mouse is an animal which has been used for many years as a suitable experimental animal in cytogenetic investigations. There is an abundance of available data, which may aid the interpretation of the results of the in vivo micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments may also be useful for the design and performance of the micronucleus test

Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 males per dose group (7 males for 1 MTD dose group)
Initial age at start of
acclimatisation: 6 - 12 weeks
Age at start of treatment: Minimum 7 weeks

Sex:

male

Details on test animals or test system and environmental conditions:

Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type II L
Bedding: Altromin saw fiber bedding (Batch: 0131)
Feed: Free access to Altromin 1324 (Batch: 0939) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.25% aqueous hydroxypropyl methylcellulose (Methocel®) as vehicle of the test item was used as negative control. All control animals were handled in an identical manner to the test group subjects.
The sampling time for the negative control was 44 h after final treatment.
Name: 0.25% aqueous hydroxypropyl methylcellulose
Route and frequency of administration: oral, twice
Volume administered: 10 mL/kg bw

Name: Aqua ad iniectabilia
Supplier: Deltamedica
Batch No.: 2006003
Physical State/Colour:liquid/colorless
Storage: ambient, at room temperature
Expiry date: May 2023
Name: hydroxypropyl methylcellulose (Methocel®)
Supplier: Colorcon
Batch No.: D180I7S002
Storage: ambient, at room temperature
Expiry date: 27 July 2023

Details on exposure:

The animals received the test item twice at a 24 h interval by the oral route. Four hours before treatment all animals were under fasting condition.

Duration of treatment / exposure:

administrations on 2 consecutive days by oral gavage.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Top dose selection is based on results of the dose range finding experiment performend as pre test using 1000, 500, and 250 mg/kg bw.

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 M

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 40 mg/kg bw
Route and frequency of
administration: ip, single
Volume administered: 10 mL/kg bw
Expiry date: October 2022

Examinations

Tissues and cell types examined:

Clinical observation, Blood cell analysis

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. The maximum dose that was applied was 1500 mg/kg bw for females and 1000 mg/kg bw for males based on data concerning toxicity of the test item provided by the sponsor and animal welfare aspects. The maximum volume which was administered was 10 mL/kg bw. The animals received the test item once.
The use of the maximum tolerated dose is generally recommended. In cases where the test item cannot be dissolved or evenly suspended in any of the well-known vehicles, the highest dose which can be formulated and administered reproducibly is used. The volume to be administered is compatible with physiological space available.
The maximum tolerated dose is defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, by inducing body weight depression or hematopoietic system cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia).
Animals treated with the highest dose (1 MTD) showed toxic effects with reduction of spontaneous activity, hunched posture, prone position, ataxia, constricted abdomen, piloerection and half eyelid closure up to 4 h after each application. 24 h after the final application all animals had fully recovered. Mice treated with 125 mg/kg bw/day (0.5 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals except for ataxia. These signs of toxicity were in total less intensely developed than in the 1 MTD group animals. 24 h after the final application all animals had fully recovered. The animals treated with 50 mg/kg bw/day (0.2 MTD) showed no signs of toxicity after the treatment with the test item.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of the peripheral blood was carried out on animals 44 h after final treatment.

METHOD OF ANALYSIS:
Cell suspensions flow cytometry

OTHER:

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Poisson-based 95% control limits

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduction of spontaneous activity, hunched posture, prone position, ataxia, constricted abdomen, piloerection and half eyelid closure up to 4 h after each application. 24 h after the final application all animals had fully recovered.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose Formulation Analysis Results
The quantification of the test item formulations showed that measured concentrations lay below the expected nominal concentrations and below the acceptance criterion. This could be due to the preparation method of the test item that resulted in a suspension of the test item with the presence of small particles. Albeit all formulations were shown to be homogenous, the presence of particles and the use of relatively small volumes for formulation analysis could be the cause for the discrepancy.
The test item formulations for the pre-experiment to determine the maximum tolerated dose and for the main experiment were prepared following the same procedure and were applied to the animals as suspensions under continuous stirring.
Additionally, the clinical signs of systemic toxicity of the animals of the highest dose group (1 MTD, 250 mg/kg) in the main experiment were consistent with the observations of the pre-experiment indicating that the doses applied in both experiments were consistent. The application of a higher dose would have led to a level of systemic toxicity contradicting animal welfare.
Thus in both experiments of this study the maximum tolerated dose of the test item was applied according to OECD 474 and the criteria for selection of the maximum dose were fulfilled and the study is considered acceptable and valid.

Bioanalysis
For analytical purposes blood samples of additional three male animals were obtained two hours after the final application of the maximum tolerated dose of the test item.
The analysis of the blood plasma showed that the test material could be detected in all samples demonstrating systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.


For further details on results incl. historical control please refer to study report attached as a confidential attachment.

Any other information on results incl. tables

Dose Group	Concentration [mg/kg bw/day]	Sampling time [h]	Rel. PCE	Proportion rel. PCE to Control [%]	MN [%]
NC	0	44	2.48	100	0.25
CPA	40	44	0.47	19.0	1.95
0.2 MTD	50	44	1.85	74.6	0.20
0.5 MTD	125	44	1.59	64.1	0.19
1 MTD	250	44	1.90	76.6	0.24

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, Art. 277455 (PY-2-B(OH)2) is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.