4-Ethoxy-2,3-difluorphenylboronic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-07-12 to 2016-12-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Dose Range Finder study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

no

Remarks:

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

EC Number:

606-729-6

Cas Number:

212386-71-5

Molecular formula:

C8 H9 B F2 O3

IUPAC Name:

(4-ethoxy-2,3-difluorophenyl)boronic acid

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

For the testing of chemicals in repeat dose oral toxicity studies in rodents, the preferred species is the rat according to guideline OECD 407 relevant for the subsequent 28-day study.
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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

Methocel® K4M Premium

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
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.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

100, 300 and 600 mg/kg: 14 consecutive days
1000 mg/kg: 7 - 9 consecutive days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 m / 5 f

Control animals:

no

Details on study design:

Dose Selection Rationale
The test item was tested for acute toxicity in Wistar rats after single dose oral administration of 300 and 2000 mg/kg. The dose of 300 mg/kg was clinically well tolerated by all rats (6 females). All (3 females) animals treated with 2000 mg/kg were killed in moribund condition approximately 3 hours after test item administration, showing the following symptoms: locomotor disturbance, piloerection, incomplete closure of the eyelids, and hunched posture. Therefore, the LD50 was considered to be between 300 and 2000 mg/kg.
In a combined Comet and Micronucleus assay with the test item, groups of 5 male SD rats were treated with 30, 100, 300 and 1000 mg/kg/d for 3 days. The rats treated with 1000 mg/kg 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 were clinically well tolerated with a slightly reduced body weight gain in 300 mg/kg animals. Histopathologic evaluation of duodenum, glandular stomach and liver revealed a minimal to mild degeneration of hepatocytes in 2 males treated with 1000 mg/kg.
The dose level of 300 mg/kg/d over a time period of 14 days was expected to cause mild to moderate systemic toxicity and 100 mg/kg/d was expected to cause no or mild systemic toxicity. Since no relevant clinical signs and/or effects on body weight were observed in groups 1 and 2 until day 14, two additional groups with 600 and 1000 mg/kg were added.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Daily

DETAILED CLINICAL OBSERVATIONS: Yes
Daily

BODY WEIGHT: Yes
The body weights for rats dosed with 100 and 300 mg/kg were were recorded before treatment and from study day 4 onwards daily. Body weight of rats dosed with >= 600 mg/kg were recorded before treatment and thereafter daily.

FOOD CONSUMPTION: Yes
Weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy and examination for gross pathological alterations were performed on all rats. Organ and tissue weights were not determined. All findings were manually recorded on a necropsy protocol. With the exception of organs designated for histopathology, the carcasses are disposed off.

HISTOPATHOLOGY: Yes
Thymus, Stomach, Small intestines, Large intestines, Ln. mesenteric

Statistics:

Since no control group was included in this study, statistical evaluation was not performed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg: Incomplete closure of the eyelids, decreased spontaneous activity, piloerection, reddish discharge from the nostrils and hypothermia (by touch) on 2 to 9 study days.
600 mg/kg: One male showed incomplete closure of the eyelids, but only for one day.
>= 300 mg/kg: Except one 300 mg/kg/d male all animals showed short-term salivation and/or rooting in the bedding material directly after treatment on several occasions. In one 1000 mg/kg male rooting in the bedding material was observed for more than 1 minute on day 7. However, short-term salivation and/or rooting in the bedding material directly after treatment indicates rather a bad taste of the test item preparation than a toxicological effect in rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

1000 mg/kg: 3 m / 3 f were sacrificed in moribund condition between day 7 and 9. Therefore, the treatment of the remaining 2 m / 2 f was stopped on day 9 and these animals were killed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight was not affected in males. On day 9 the surviving females showed a slightly increased mean body weight compared with 300 and 100 mg/kg females. Except for 1000 mg/kg males, which sowed a slightly decreased mean body weight gain from day 1 to 9, mean body weight gain increased mostly dose-dependently.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly increased in 1000 mg/kg males and reduced in 1000 mg/kg females from day 1-7 compared to 600, 300 and 100 mg/kg/d group animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg: All rats showed a massively increased filling (food) of the stomach. One female had multifocal black discolorations in the pylorus mucosa. All males and 3/5 females revealed an obstipation of the colon in addition.
600 mg/kg: All rats of both genders revealed a moderately filling (food) of the stomach, which was clearly more than normally seen in control rats.
300 mg/kg: One male showed multifocal brown discolorations in the thymus. Since the histopathological correlate (hemorrhage, acute, multifocal) was considered to be spontaneous in nature, the multifocal brown discolorations in the thymus is not considered treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

>= 300 mg/kg: Histopathology of the selected organs did not reveal any treatment-related lesions.
Especially at 1000 and 600 mg/kg no lesions were seen that could explain the pathogenesis of the macroscopic gastro-intestinal findings (moderately to massively increased filling of the stomach with food, obstipation). Most likely, the massively increased filling of the stomach led to an increased intra-abdominal pressure. Increased abdominal pressure might have led to a general anoxia by respiratory embarrassment and to marked effects on the hemodynamics of the abdominal viscera leading to the reduced general condition and premature sacrifice of the animals dosed with 1000 mg/kg. The origin for the massive food intake itself remains unclear, maybe a bad taste of the test item might have contributed to the marked intake of food.
Histopathological findings in the thymus and mesenteric lymph nodes were considered to be spontaneous in nature.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Daily oral dosing via gavage with 1000 mg/kg of the test item was not tolerated in male and female Wistar (Han) rats due to severe clinical symptoms leading to premature sacrifice. The massively increased filling (food) of the stomach observed at necropsy had no histomorphological correlate, but might be the primary reason for the reduced general condition and premature sacrifice of the animals.
The dosing with 600, 300 and 100 mg/kg was clinically tolerated for 14 days without histopathological findings in the selected organs. However, a moderately increased filling (food) of the stomach was still noted at 600 mg/kg. Therefore, the dose level of 600 mg/kg is considered suitable to be used as high dose in subsequent repeat-dose toxicity studies of 28 days duration or longer.

Executive summary:

Objective

This 14 -day dose range finding study was performed to investigate the clinical tolerability of the anticipated dose levels for a subsequent 28 -day study according to OECD 407 in rats.

Study Design

The test item was administered once daily via gavage on 7 days per week for 14 days to 4 groups of male and female Crl:WI (Han) rats at doses of 100, 300, 600 or 1000 mg/kg. Due to animal welfare reasons the dosing with 1000 mg/kg was stopped on study day 9. The rats were gang-housed under conventional conditions. Each dose group consisted of 5 male and 5 female rats, i.e. 40 rats in total were used.

Appearance, behaviour and mortality were monitored daily and feed consumption weekly. The body weights for rats dosed with 100 and 300 mg/kg were determined on day 1 from study day 4 onwards daily and daily for rats dosed with 600 and 1000 mg/kg.

At the end of the treatment period, all animals were subjected to a necropsy and examination for gross pathological alterations was performed on all rats including those that were killed due to animal welfare reasons.

Results

Six rats (3 m / 3 f) dosed with 1000 mg/kg were sacrificed in moribund condition between day 7 and 9. Therefore, the treatment of the remaining animals (2 m / 2 f) was stopped on day 9 and these animals were killed. All rats treated with 600, 300 and 100 mg/kg survived until their scheduled sacrifice.

Treatment-related clinical signs such as incomplete closure of the eyelids, decreased spontaneous activity, piloerection, reddish discharge from the nostrils and hypothermia (by touch) were observed at 1000 mg/kg on 2 to 9 study days. At 600 mg/kg one male showed incomplete closure of the eyelids, but only for one day. Except one male rat dosed with 300 mg/kg, all animals treated with >= 300 mg/kg showed short-term salivation and/or rooting in the bedding material directly after treatment on several occasions. In one male rat dosed with 1000 mg/kg, rooting in the bedding material was observed for more than 1 minute on day 7. However, short-term salivation and/or rooting in the bedding material directly after treatment indicates rather a bad taste of the test item preparation than a toxicological effect in rats.

Mean body weight was not affected in males. On day 9 the surviving females showed a slightly increased mean body weight compared with 300 and 100 mg/kg females. Except for 1000 mg/kg males, which sowed a slightly decreased mean body weight gain from day 1 to 9, mean body weight gain increased mostly dose-dependently.

Food consumption was slightly increased in 1000 mg/kg males and reduced in 1000 mg/kg females from day 1 -7 compared to 600, 300 and 100 mg/kg group animals.

Gross pathology revealed treatment-related lesions at >= 600 mg/kg. At 1000 mg/kg a massive filling (food) of the stomach was observed in all males and females. One of the 1000 mg/kg female had multifocal black discolorations in the pylorus mucosa without showing a histomorphological correlate. In addition all males and 3/5 females revealed an obstipation of the colon. At 600 mg/kg, all rats of both sexes showed a moderately filled stomach.

Histopathology revealed neither treatment-related lesions in the selected organs nor lesions that might explain the increased stomach filling at 1000 and 600 mg/kg. At 1000 mg/kg the massive filling of the stomach most likely, led to an increased intra-abdominal pressure with general anoxia by respiratory embarrassment and to marked effects on the hemodynamics of the abdominal viscera which finally led to the reduced general condition and premature sacrifice of the animals.

Conclusion

Daily oral treatment with 1000 mg/kg of the test item was not tolerated in male and female Wistar (Han) rats due to severe clinical symptoms leading to premature sacrifice. The massively increased filling (food) of the stomach observed at necropsy had no histomorphological correlate, but might be the primary reason for the reduced general condition and premature sacrifice of the animals. The doses of 600, 300 and 100 mg/kg were clinically tolerated for 14 days without histopathological findings in the selected organs. However, a moderately increased filling (food) of the stomach was still noted at 600 mg/kg. Therefore, the dose level of 600 mg/kg is considered suitable to be used as high dose in subsequent repeat-dose toxicity studies of 28 days duration or longer.