(S)-5-fluoro-3-methylisobenzofuran-1(3H)-one

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:

07 March 2016 to 15 March 2016

Reliability:

1 (reliable without restriction)

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:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for Testing of Chemicals, Guideline 407: "Repeated Dose 28-day Oral Toxicity Study in Rodents", Paris, 03 October 2008.

Deviations:

no

Principles of method if other than guideline:

The type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee (DEC 14-59) as required by the Dutch Act on Animal Experimentation (February 1997).

Analysis of test item in vehicle for concentration, stability, homogeneity were not performed, however, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 4 hours after adding vehicle to the test item

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3S)-5-fluoro-3-methyl-1,3-dihydro-2-benzofuran-1-one

EC Number:

943-180-4

Cas Number:

1803573-19-4

Molecular formula:

C9H7FO2

IUPAC Name:

(3S)-5-fluoro-3-methyl-1,3-dihydro-2-benzofuran-1-one

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Purity: 100 % (HLPC %area)

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Remarks:

outbred, SPF-Quality

Details on species / strain selection:

Rationale: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Source: Charles River Deutschland, Sulzfeld, Germany.

Sex:

male/female

Details on test animals or test system and environmental conditions:

6.4. Test System
Total number of Animals: 12 males and 12 females (females were nulliparous and nonanimals pregnant).
Age at start of treatment: Approximately 9 weeks.
Identification: Tailmark (markerpen) and earmark.
Randomization: By computer-generated random algorithm according to body weight,with all animals within ± 20% of the
sex mean.
Acclimatization period: At least 5 days before the start of treatment under laboratory conditions.
Health inspection: Upon receipt of the animals.

6.6. Animal Husbandry
Room number: Room A0.61b.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air
changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and
had no effect on the outcome of the study.

Accommodation: Group housing of 3 animals per sex in Makrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

Diet Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036.

Details on oral exposure:

6.7. Treatment
Method: Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
A dose control system (DCS) was used to verify the dosing procedure on Days 3 to 7.dose
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to body weights determined on Days 1 and 4.
Frequency: Once daily for up to 7 consecutive days, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A dose control system (DCS) was used to verify the dosing procedure on Days 3 to 7.

Duration of treatment / exposure:

7 consecutive days

Frequency of treatment:

Daily

No. of animals per sex per dose:

3 males or 3 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Male and female Wistar Han rats, approximately 9 weeks of age on study Day 1, were administered PF-06811569 via oral gavage daily for at least 7 consecutive days as indicated in the following table.

Group Dose level Number of animals Animal numbers
Number (mg/kg/day) Males Females Males Females
1 0 (vehicle)a 3 3 01-03 13-15
2 150 3 3 04-06 16-18
3 300 3 3 07-09 19-21
4 1000 3 3 10-12 22-24
a The vehicle was propylene glycol, specific gravity 1.036.

Positive control:

Shown as Group 1 above.

Examinations

Observations and examinations performed and frequency:

6.8. Observations
Mortality / Viability: At least twice daily.

Clinical signs: On Day 1, detailed clinical observations were made once, from Day 2 onwards, these observations were made at least immediately (0- 30 min) and 2 hours (± 30 min) after dosing. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades are reported, as well as the percentage of animals affected in summary tables.

Body weights On Days 1, 4 and 7.

Food consumption Over Days 1-4 and 4-7.

Water consumption Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Sacrifice and pathology:

6.10. Pathology
6.10.1. Necropsy
Animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). The animals were subsequently exsanguinated and subjected to a full post mortem examination. All animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

Identification marks: not processed Ovaries
Adrenal glands 4 Spleen
Brain [cerebellum, mid-brain, cortex] (7 Stomach
levels)
Cervix Testes*
Epididymides * Thymus
Heart Uterus
Kidneys Vagina
Liver All gross lesions
* Fixed in modified Davidson's solution, prepared at Charles River Den Bosch using Formaldehyde 37-40%,
Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation,
Bedford, USA). Tissues were transferred to formalin after fixation for at least 24 hours.

6.10.2. Organ Weights
The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus
Liver

6.10.3. Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed,
embedded in paraffin wax (Klinipath, Duiven, The Netherlands) and cut at a thickness of 2-4
micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

6.10.4. Histopathology
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- thymus, liver and spleen of all males and females of Groups 2 and 3 and kidneys of all males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4,
- all gross lesions.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Histopathology was subjected to a peer review.

6.11. Interpretation
A descriptive statistical analysis was performed (mean, SD and/or median).

Other examinations:

6.9. Clinical Laboratory Investigations
Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.38 a.m. (performed as a separate procedure and not part of the necropsy procedure, see deviation). Animals were deprived of food overnight (for a maximum of 24 hours), but water was available. Blood samples were drawn from the retroorbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 1 the animals were observed at least once for detailed clinical signs. Based on the results it was decided to document two observations (t=0 and t=2 hours after dosing) for Day 2 onwards, therefore two tables of clinical signs are presented.

Lethargy was noted for males and females treated at 1000 mg/kg throughout the observation period. The animals treated at 150 and 300 mg/kg showed lethargy on Days 1 and/or 2. Watery discharge from the eyes, piloerection and/or ptosis were noted for the experimental animals of both sexes on Days 1 and/or 2 in a dose-related trend.

Salivation seen after dosing among the animals of all dose groups in a dose-related manner was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

All males at 1000 mg/kg showed slight weight loss (up to 3%) between Days 1-4 with subsequent weight gain between Days 4-7. Mean body weights of these males was lower than controls throughout treatment.

All females at 150, 300 and 1000 mg/kg showed slight weight loss (up to 3%) between Days 1-4 with subsequent weight gain between Days 4-7. Mean body weights of these females was lower than controls throughout treatment, however no dose related response was noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced food consumption (absolute and relative) was noted for both sexes at 1000 mg/kg, which partially recovered during treatment. These changes generally showed an apparent dose-related trend.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematocrit and mean corpuscular volume (MCV) in males and reticulocytes in females and platelet count in males and females showed a dose-related increase over the dose groups.
One female at 1000 mg/kg showed higher relative neutrophil counts with concurrently reduced relative lymphocyte counts. This shift in type of white blood cells in only one female was considered to be a secondary non-specific response to stress and to be of no toxicological significance.
Any other changes in haematology parameters were considered to be unrelated to treatment as
these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following dose related changes in clinical biochemistry parameters (generally) occurred
with an apparent dose-related trend
- Higher total protein in both sexes.
- Lower alkaline phosphatase in females
- Higher albumin in both sexes.
- Higher triglyceride in females.
- Higher alanine aminotransferase (ALAT) activity in both sexes.
- Higher creatinine in males.
Other clinical biochemistry parameters were not considered to have been affected by
treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was an apparent test item-related increase in:
- Liver weight in males at 300 and 1000 mg/kg and in females at 300 and 1000 mg/kg (up to 63% absolute and 78% relative in males and 72% absolute and 80% relative in females).
- Kidney weight in males at 300 and 1000 mg/kg (up to 20% absolute and 31% relative).
There was an apparent test item-related decrease in:
- Thymus weight in males at 300 and 1000 mg/kg and in females at 150, 300 and 1000 mg/kg (up to -44% absolute and -39% relative in males and -47% absolute and -45% relative).
- Spleen weight in males at 300 and 1000 mg/kg (up to -26% absolute and -19% relative). There were no other test item-related organ weight changes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with PF-06811569 were noted in the liver, thymus, spleen and/or kidneys of males and females.
- Liver, hepatocellular hypertrophy (centrilobular) was present in males and females starting at 300 mg/kg up to slight degree.
- Kidneys, hyaline droplet accumulation was present in males starting at 150 mg/kg up to marked degree.
- Thymus, lymphoid atrophy was present in males at 1000 mg/kg and in females starting at 300 mg/kg up to slight degree.
- Spleen, lymphoid atrophy was present in males and females at 1000 mg/kg up to slight degree.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Wistar rats were treated with PF-06811569 for 7 consecutive days by daily oral gavage at dose levels of 150, 300 and 1000 mg/kg.

All animals survived up to the scheduled day of necropsy. Lethargy was noted for males and females treated at 1000 mg/kg throughout the observation period. The animals treated at 150 and 300 mg/kg showed lethargy on Days 1 and/or 2. Watery discharge from the eyes, piloerection and/or ptosis were noted for the experimental animals of both sexes on Days 1 and/or 2 in a dose-related trend.

Body weight loss over Days 1-4 was noted in males and females predominantly at the highest dose and was accompanied by reduced food consumption. Since body weight gain was noted after Day 4, these findings were not considered adverse.

Histopathological examination revealed non-adverse changes in liver, thymus, spleen and kidneys:
Hepatocellular hypertrophy in the liver was noted in both sexes at 300 and 1000 mg/kg. In the absence of any other indicator of hepatocellular toxicity this histopathological finding was not considered adverse (Ref 1.). However, the increased liver weights recorded in males at 300 and 1000 mg/kg (relative to body weight +36% and +78% resp.) and females at 300 and 1000 mg/kg (relative to body weight +80%) was considered adverse at these percentages (Ref 2.).

Lymphoid atrophy was noted in the thymus of the 300 and 1000 mg/kg and in the spleen of the 1000 mg/kg treated rats and was not accompanied by any other indicator of toxicity and was, at this degree, considered non-adverse.

Hyaline droplet accumulation was recorded in the male kidneys starting at 150 mg/kg, which was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref 3.). The increased hyaline droplet accumulation in the male kidneys was accompanied by an increased kidney weight of 20 and 31% relative to body weight at 300 and 1000 mg/kg and was not accompanied by indicators of tubular damage such as granular casts or increased tubular basophilia therefore this was not considered to be adverse.

The slight changes in haematology (e.g. haematocrit, mean corpuscular volume, reticulocytes and platelet count) and clinical biochemistry (total protein, alkaline phosphatase, albumin, triglyceride, alanine aminotransferase activity and creatinine). Based on the slight nature these findings these changes were not considered to be adverse.

Based on the higher liver weight presented in this report, PF-06811569 is considered to have
toxicological potential starting at 300 mg/kg.