4-Ethoxy-2,3-difluorphenylboronic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay (OECD 471) – Screening Test

The mutagenic potential of the test item was examined in a screening assay similar to OECD TG 471 using Salmonella typhimurium tester strains TA98, TA100 and E. coli WP2 uvrA.

Precipitation of the test item on the agar plates occurred at concentrations of >= 1580 μg/plate and toxicity to bacteria at 5000 µg/plate. The treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test item treatments in the absence and presence of S9, the test item was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA.

 

In vitro MNT Assay (OECD 487) – Screening Test

The mutagenic potential of the test item was examined in a screening assay equivalent to OECD Guideline 487 using TK6 cells both with and without addition of S9 mix.

Precipitation until the end of exposure was observed at >= 2476 μM. Excessive cytotoxicity was observed at concentrations of >= 619 μM (-S9 mix) or 1238 μM (+S9 mix), respectively. In the absence of metabolic activation, the test item showed relevant, concentration-dependent induction of micronucleus frequencies in the non-cytotoxic concentration. In the presence of metabolic activation, the test item showed no relevant induction of micronucleus frequencies up to the limit of cytotoxicity.

The test item was mutagenic in this screening test system in the absence of metabolic activation (S9 mix).

 

Overall Conclusion

Since the test item was mutagenic (with and without S9 mix) in the Ames Screening Test and induced cytogenic damage in the in vitro MNT Screening test, in vivo studies are required to met a final conclusion on classification and labelling.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Screening assay (only results were reported, limited number of strains, methodological details are missing)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

no

Remarks:

Screening test

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

1st series (TA 98, TA 100, E. coli WP2 uvr A): 5, 15.8, 50, 158, 500, 500, 1580 and 5000 μg/plate
2nd series (TA 100, E. coli WP2 uvr A): 500, 889, 1580, and 2810 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: daunomycin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies.

Rationale for test conditions:

Screening assay

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduced background lawn at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation at the beginning of the experiment starting at 1580 µg/plate.

Ames test:
- Signs of toxicity : Reduced bacterial background lawn at 5000 µg/plate in all three strains.

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA under the experimental conditions described.

Executive summary:

Study design

The mutagenic potential of the test item was examined in a screening assay equivalent to OECD GL 471 using Salmonella typhimurium tester strains TA 98, TA 100 and Escherichia coli WP2 uvrA. The plate incorporation test (two parallel plates per condition) with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. The test item was dissolved in DMSO and tested at concentrations ranging from 5 - 5000 μg/plate.

Results

Precipitation of the test item on the agar plates occurred at concentrations of >= 1580 μg/plate and toxicity to bacteria at 5000 µg/plate. The treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following test item treatments in the absence and presence of S9, the test item was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test item was mutagenic in Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Screening assay (methodological details are missing)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

not applicable

GLP compliance:

no

Remarks:

Screening test

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

not further specified

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

77.2 - 9903 µM

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: griseofulvin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

Exposure
-S9 mix: 23 h
+S9 mix: 3 h

Rationale for test conditions:

Screening assay

Statistics:

n.a.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: until the end of exposure from 2476 µM onwards

STUDY RESULTS
- Concurrent vehicle negative and positive control data


Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o When cytokinesis block is not used: RICC, RPD or PD, as well as the number of cells treated and of cells harvested for each culture : Please refer to "Summary Tables" as attached background material

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate : Please refer to "Summary Tables" as attached background material

Conclusions:

The test item was mutagenic in this screening test system in the absence of metabolic activation (S9 mix).

Executive summary:

Study design

The mutagenic potential of the test item was examined in a screening assay equivalent to OECD Guideline 487 using TK6 cells both with and without addition of S9 mix. The test item was dissolved in DMSO and tested at concentrations ranging from 77.2 - 9903 µM.

Results

Precipitation until the end of exposure was observed at >= 2476 μM. Excessive cytotoxicity was observed at concentrations of >= 619 μM (-S9 mix) or 1238 μM (+S9 mix), respectively.

In the absence of metabolic activation, the test item showed relevant, concentration-dependent induction of micronucleus frequencies in the non-cytotoxic concentration.

In the presence of metabolic activation, the test item showed no relevant induction of micronucleus frequencies up to the limit of cytotoxicity.

Conclusion

The test item was mutagenic in this screening test system in the absence of metabolic activation (S9 mix).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Combined in vivo Comet Assay and in vivo Micronucleus Assay (OECD 489/OECD 474)

The combined study was conducted to determine the genotoxic potential of the test item by detecting DNA damage in tissues of liver, glandular stomach, duodenum and cytogentic damage in bone marrow.

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.

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.

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.

To investigate the equivocal result in the bone marrow of treated animals, a further in vivo MNT Assay was performed.

In vivo MNT Assay (OECD 474)

The objective of this study was to investigate the potential of the test item to induce micronuclei formation in the bone marrow of rats.

The current micronucleus test utilized femurs from male and female animals from a 14-day oral dose range finding toxicity study in rats. Both femurs were taken from animals treated in that study, 24 hours after the last administration, processed and evaluated microscopically as in a standard acute micronucleus study. The test item was given orally by gavage at doses of 100, 300 or 600 mg/kg body weight.

No relevant increases in the number of MN-PCE were observed in any of the female or male groups treated with the test item compared to the historical negative controls (males: 1.7 +/- 0.7 ‰; females: 1.3 +/- 0.7 ‰).

Therefore, the test item is not considered to be clastogenic or aneugenic in the micronucleus test in bone marrow of male and female rats.

Overall Conclusion

Based on the results of the Combined in vivo Comet Assay and in vivo Micronucleus Assay (OECD 489/OECD 474) the test item is not considered to be genotoxic in liver and glandular stomach. Since an equivocal result in bone marrow of treated animals at toxic dose was oberserved, a further in vivo MNT Assay according to OECD 474 was performed. In the second in vivo MNT Assay no relevant increases in the number of MN-PCE were observed.

Therefore, the test item did not show a clastogenic and aneugenic effect in bone marrow of rats.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

09-2014

Deviations:

no

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

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

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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.

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

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

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.

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.

Executive summary:

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-27 to 2016-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

14-day dose range finder

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH SR2(R1)

Version / remarks:

2011

Deviations:

not applicable

GLP compliance:

no

Remarks:

This study was conducted and reported under GRP (Good Research Practices) conditions. GRP is a voluntary internal Quality Management System implemented in Merck Biopharma/EMD Serono R&D for all non-GxP regulated activities.

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Crl: WI (Han) rat is widely accepted by Health Authorities as an appropriate experimental model, with documented susceptibility to a wide range of toxic substances.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 153 - 253 g
- Fasting period before study: not specified
- Housing: gang-housed in type IV Makrolon cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.5
- Humidity (%): 48.6 - 78.6
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From day 1 - 15

Route of administration:

oral: gavage

Vehicle:

Methocel® K4M Premium

Details on exposure:

For preparation the appropriate amount of the test item was suspended in 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium). Stability and homogeneity data revealed that the preparation is homogeneous and stable for at least 3 days at room temperature. However, as the recoveries from nominal were improved on days 2 and 3, the test item formulation was prepared on the day before usage and stirred overnight. A determination of the test item concentrations in the vehicle was not performed. The volume of administration was 5 mL/kg bw.

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

once daily

Post exposure period:

no

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 m / 5 f

Control animals:

no

Positive control(s):

In this study, no animals were treated with a positive control. However, positive control slides from a previous study in which a single oral dose of 10 mg cyclophosphamide/kg bw was administered to male and female rats were coded together with the slides of the current study in order to check accurate slide reading. These slides were introduced as the positive control group during evaluation.

Tissues and cell types examined:

Bone marrow cells from male and female rats (Crl: Wistar Han) used in a 14-day range finding study

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In an acute oral toxicity study, the LD50 was determined to be between 300 and 2000 mg/kg after single oral administration. In a combined Comet and MNT Assay the rats treated with 1000 mg/kg bw/d showed lethargy, irregular breathing, porphyrin staining, partially closed eyelids and body weight loss between 1.5 and 9.8%. 30, 100 and 300 mg/kg bw/d were clinically well tolerated with a slightly reduced body weight gain in 300 mg/kg bw/d animals. Histopathologic evaluation of duodenum, glandular stomach and liver revealed a minimal to midl degeneration of hepatocytes in 2 males treated with 1000 mg/kg bw/d.
Therefore, it was expected that the high dose level of 300 mg/kg bw/d over a time period of 14 days will cause mild to moderate systemic toxicity, the low dose level of 100 mg/kg is expected to cause no or mild systemic toxicity. However, animals treated with 300 mg/kg bw/d did not show any clinical signs indicative for toxicity, therefore, two additional dose groups (males and females) treated with 600 mg/kg bw/d and 1000 mg/kg bw/d were introduced into the study. All animals of the 1000 mg/kg bw/d had to be killed prematurely, hence, 600 mg/kg bw/d administered for 14 days were considered to be the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
From all surviving animals, both femurs were taken during necropsy. Necropsy was performed 24 hours after the last treatment. lmmediately after the rats were killed, the femurs of each animal were dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum with the aid of a syringe, and suspended in serum. This suspension was filtered through cellulose and centrifuged for 5 min at 201*g. The cell pellet was then resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension. For each animal, three slides were prepared. After drying, the slides were stained according to a modified Giemsa-staining method using Giemsa's solution with Weise buffer solution and mounted in Entellan.

Quality Control of Microscopic Slides
For each animal, three slides were prepared. After a quality check assessing density and staining of the slides, one out of three slides was selected for further investigation and coded prior to microscopic evaluation. The code numbers were generated using the IKG Random randomisation program.

METHOD OF ANALYSIS:
Evaluation of Slides and Scoring of Micronuclei-Containing Cells
A total of 4000 PCE per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250 x). Round particles of about 1/20 - 1/5 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. They were differentiated from granules by thorough examination at different plane levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic.
For determination of bone marrow toxicity, both erythrocyte stages (PCE and NCE) were counted separately up to a total of 1000 erythrocytes per animal and the proportion of PCE among total erythrocytes was determined (%PCE). Then counting was limited to PCE.
NCE with micronuclei, however, observed during scoring of polychromatic erythrocytes were registered also.
When analysing samples, the proportion of polychromatic (immature) erythrocytes among total erythrocytes in treated animals should not be less than 20% of the concurrent vehicle control proportion.

Evaluation criteria:

The study is acceptable as valid, if:
-the concurrent positive control or scoring control should induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared to the concurrent negative control and
-the appropriate number of cells has been analysed.

The principal parameter for evaluation of the results of this test system is the proportion of micronuclei-containing polychromatic erythrocytes (MN-PCE). This proportion is increased by treatment of animals with mutagenic test items as compared to the respective negative control.

A test item is considered clearly positive if the acceptability criteria are fulfilled and
-any increase in mean micronucleus frequency observed is outside the distribution of the historical negative control data (95% control limits) and
-the increase is dose related or occurs at the highest dose level and
-the mean percentage of PCE in the treated group is not less than 20% of the mean percentage of PCE at the concurrent negative control group.

A test item is considered clearly negative, if in all experimental conditions examined
-all results are inside the distribution of the historical negative control data and
-bone marrow exposure to the test item occurred.

Statistics:

The numerical calculations were performed using validated Excel® spreadsheets.
As no concurrent vehicle control group was included in this study, group mean values were compared to the historical control data. No statistical tests were performed.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

According to the predetermined criteria for the interpretation of results, the test item was not mutagenic in the micronucleus test in bone marrow of male and female rats treated with a daily oral dose of 100, 300 or 600 mg/kg for 14 days under the conditions described.

Executive summary:

Objective

The objective of this study was to investigate the potential of the test item to induce micronuclei formation in the bone marrow of rats.

Study Design

The current micronucleus test utilized femurs from male and female animals from a 14-day oral dose range finding toxicity study in rats. Both femurs were taken from animals treated in that study, 24 hours after the last administration, processed and evaluated microscopically as in a standard acute micronucleus study. The test item was given orally by gavage at doses of 100, 300 or 600 mg/kg body weight. No animals in this study were treated with a negative or positive control compound. However, positive control slides from a previous study were coded together with the slides of this study in order to check accurate slide reading.

Bone marrow smears were prepared from two femurs of each animal and stained with Giemsa's solution. A total of 30 animals (5 for each main group and sex) were used for slide preparation. For microscopic investigation from five animals per group (30 animals in total) one out of three slides prepared from each animal was selected and coded. The number of polychromatic (immature) erythrocytes (PCE) containing micronuclei (MN-PCE) per 4000 polychromatic erythrocytes was determined.

The proportion of PCE among total erythrocytes was determined based on the analysis of 1000 erythrocytes per animal. The micronucleated nonnochromatic erythrocytes (NCE) were registered also when scoring the polychromatic erythrocytes.

Results

The slide reading control (positive control slides of male and female rats treated with 10 mg/kg cyclophosphamide) showed the expected increase in the number of MN-PCE. The values obtained were perfectly within the historical positive control (males: 16.4 +/- 5.8 ‰; females: 10.7 +/- 3.9 ‰). For each animal, the appropriate number of cells (4000 PCE) was analysed. Hence, the study was accepted as valid.

In the 14 -day oral dose range finding toxicity, the daily dosing with 600 mg/kg for 14 days was considered to be the maximum tolerated dose and therefore appropriate for micronucleus investigations.

In none of the groups treated with the test item, the mean percentage of PCE was less than 20 % of the historical vehicle control (males: 49.5 +/- 5.27 ‰; females: 43.9 +/- 9.72 ‰), indicating that no relevant bone marrow toxicity was present.

No relevant increases in the number of MN-PCE were observed in any of the female or male groups treated with the test item compared to the historical negative controls (males: 1.7 +/- 0.7 ‰; females: 1.3 +/- 0.7 ‰).

Conclusion

According to the predetermined criteria for the interpretation of results, the test item was not mutagenic in the micronucleus test in bone marrow of male and female rats treated with a daily oral dose of 100, 300 or 600 mg/kg for 14 days under the conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Although mutagenic and cytogenic effects were observed in vitro, the test item did not induce DNA damage in vivo in selected tissues and did not show a clastogenic and aneugenic effect in bone marrow of rats. Based on the in vivo results, the test item is not considered to be mutagenic or clastogenic/aneugenic.

Justification for classification or non-classification

Based on the results, the test item is not classified for genotoxicity according to Regulation (EC) No 1272/2008.