3-chloro-4-fluoroaniline

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th August 2012 to 22nd October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-chloro-4-fluoroaniline

EC Number:

206-682-8

EC Name:

3-chloro-4-fluoroaniline

Cas Number:

367-21-5

Molecular formula:

C6H5ClFN

IUPAC Name:

3-chloro-4-fluoroaniline

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- 20 males, 20 females (females were nulliparous and non-pregnant).
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: Males - 145-148g. Females - 124-126g
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage- enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food.
- Water: Free access to tap water except during motor activity measurements.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions

DETAILS OF FOOD AND WATER QUALITY: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 8 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 24th September 2012 To: 21st October 2012

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

propylene glycol

Remarks:

Propylene glycol (Merck, Darmstadt, Germany), specific gravity 1.036

Details on oral exposure:

Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 Days. Animals were dosed up to the day prior to necropsy.

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw (total dose)

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent no treatment

Details on study design:

Rationale for dose levels
Based on the results of a 10-day range finding study, the dose levels for this 28-day oral gavage study were selected to be 0, 5, 15 and 50 mg/kg.

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals this were also performed outside the home cage in a standard arena. Clinical observations were conducted between 1 and 2 hours after dosing (except for the last observations in a standard arena) which was considered to be the peak period of anticipated effects after dosing based on results of the dose range finding study. 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) were scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected.
Functional Observations: During week 4 of treatment, the following tests were performed on all animals.
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head. Functional observations commenced between 1 and 2 hours after dosing which was considered to be the peak period of anticipated effects after dosing based on results of the dose range finding study.
Body weights Weekly.
Food consumption Weekly.
Water consumption Subjective appraisal was maintained during the study and a quantitative assessment introduced in the event of a suspected treatment related effect.

Sacrifice and pathology:

Necropsy
Animals were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. 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.
Ovaries, Adrenal glands, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, Prostate gland, Rectum, Colon, Duodenum, Sciatic nerve, Epididymides, Seminal vesicles including coagulating gland, Eyes (including optic nerve and harderian gland), Skeletal muscle, Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum Testes, Kidneys,
Thymus, Thyroid including parathyroid, Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, Vagina,.

Histopathology
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all group 1 and 4 animals,
- spleen of all animals of groups 2 and 3, based on (possible) treatment-related changes in this organ 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Calm behavior was observed for all animals at 50 mg/kg from Day 23 onwards and for all animals at 15 mg/kg on Days 27 and 28.
No clinical signs of toxicity were noted for control animals and animals at 5 mg/kg during the observation period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Higher white blood cell counts in males at 50 mg/kg.
- Lower red blood cell counts in females at 15 mg/kg and males and females at 50 mg/kg.
- Higher relative reticulocyte counts in males and females at 15 and 50 mg/kg (without statistical significance for males at 50 mg/kg).
- Higher red blood cell distribution width (RDW) values in females at 15 mg/kg and males and females at 50 mg/kg.
- Lower haemoglobin levels in males at 15 mg/kg and males and females at 50 mg/kg.
- Higher mean corpuscular volume (MCV) values in males and females at 50 mg/kg.
- Higher mean corpuscular haemoglobin (MCH) values in males and females at 50 mg/kg.
- Lower mean corpuscular haemoglobin concentration (MCHC) in females at 50 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Higher total bilirubin levels in males and females at 50 mg/kg. Males and females at 15 mg/kg showed slightly higher total bilirubin levels without statistical significance.
- Higher alanine aminotransferase (ALAT) levels in females at 50 mg/kg.
- Higher potassium levels in males (without statistical significance) and females at 50 mg/kg.
- Lower chloride levels in females at 50 mg/kg.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Females at 15 mg/kg and males and females at 50 mg/kg showed a lower motor activity when compared to control animals as was shown by lower total movements and lower ambulations (with statistical significance for females). Motor activity was similar between control animals and males and females at 5 mg/kg and males at 15 mg/kg.
All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Spleen weights and spleen to body weight ratios of males and females at 50 mg/kg were markedly higher compared to those of control animals. In addition, liver weights and liver to body weight ratios of males and females at 50 mg/kg were higher compared to those of control animals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Spleens of all males and all females at 50 mg/kg appeared enlarged at necropsy, whereas spleens of only one control male, one male at 5 mg/kg and one male and one female at 15 mg/kg also appeared enlarged at necropsy.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation of the right kidney, a reddish hard nodule on the caecum, dark foci on the thymus, reddish foci on the lungs, yellowish soft nodules on the head or tail of the left epididymis, a reduced size of the left seminal vesicle, presence of fluid in the uterus, tan foci on the clitoral glands and watery-clear cysts on the uterus.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related microscopic findings consisted of an increase in hemopoietic foci, primarily erythroid and an increase in hemosiderin pigment in males and females at 15 and 50 mg/kg.
Hemopoietic foci increased in severity from minimal in controls to moderate in males at 15 and 50 mg/kg and females at 50 mg/kg with incidence rising from four in control males, one in control females to all five animals of both sexes at 15 and 50 mg/kg. This was statistically significant (p < 0.05) in males at 50 mg/kg and in females at 15 and 50 mg/kg.
Hemosiderin pigment at minimal or slight degree increased in incidence from none in control males to all five males at 50 mg/kg and in females from three in the control group to all five in groups at 15 and 50 mg/kg. This was statistically significant (p < 0.01) in males at 50 mg/kg.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw (total dose)

System:

immune system

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw (total dose)

System:

gastrointestinal tract

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

Based on the effects indicative of haemolytic aenemia and extramedullary haemopoiesis at dose levels of 15 and 50 mg/kg, a No Observed Adverse Effect Level (NOAEL) for PF-01458762 of 5 mg/kg was established.

Executive summary:

Title
Repeated dose 28-day oral toxicity study with PF-01458762 by daily gavage in the rat.
Guidelines
The study was based on the following guidelines.
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
- OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.
Rationale for dose levels
Based on the results of a 10-day range finding study, the dose levels for this 28-day oral gavage study were selected to be 0, 5, 15 and 50 mg/kg.
Study outline
The test substance, formulated in propylene glycol, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.
Evaluated parameters
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.
The following parameters were evaluated: clinical signs daily; functional observation tests in week 4 body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.
Results
Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours.
No mortality occurred during the study period, but during the last week of treatment, calm behaviour was observed for animals at 15 and 50 mg/kg which was also shown by a lower motor activity.
Treatment with PF-01458762 caused haemolytic aenemia and extramedullary haemopoiesis at 15 mg/kg and more severely at 50 mg/kg. These effects were reflected by the following changes in haematology and clinical biochemistry parameters: lower red blood cell counts, higher relative reticulocyte counts, higher red blood cell distribution width (RDW) values, lower haemoglobin levels, higher mean corpuscular volume (MCV) values, higher mean corpuscular haemoglobin (MCH) values, lower mean corpuscular haemoglobin concentration (MCHC), higher potassium levels and higher total bilirubin levels. Moreover, enlarged spleens and markedly higher spleen weights were observed and accompanied by microscopic findings consisting of an increase in hemopoietic foci, primarily erythroid, and an increase in hemosiderin pigment. Animals at 50 mg/kg showed also higher liver weights which was accompanied by higher white blood cell counts, higher alanine aminotransferase (ALAT) levels and/or lower chloride levels, but these findings were not supported by histopathological evidence of organ dysfunction. 
No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. body weight, food consumption, hearing ability, pupillary reflex, static righting reflex and grip strength).
Conclusion
Based on the effects indicative of haemolytic aenemia and extramedullary haemopoiesis at dose levels of 15 and 50 mg/kg, a No Observed Adverse Effect Level (NOAEL) for PF-01458762 of 5 mg/kg was established.