9917E

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study using standard method; large dosing factor

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Annex V

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

yellow powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd, UK
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: males: 185-232 g; females: 151-187 g
- Fasting period before study:
- Housing: groups of - Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2 C
- Humidity (%): 55+/- 15 %
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method of administration:
Gavage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Method: HPLC with UV detection (350 nm)
Column: Luna 5µm C18 (250X4.6 mm id
Solvent A: acetonitrile
Solvent B: 0,1 M ammonium acetate in water with 1% acetonitrile
Gradient: 100 % B - 90% B in 16 minutes

Results:
Stability: Samples were stored for 14 days in the dark; 96 -100 % of initial levels were found
Homogeneity: triplicate determinations for all dose levels showed good homogeneity
Dosage/Concentration: Weekly determinations in samples of all dose levels gave measured concentrations of 94 -110 % of nominal values.

Duration of treatment / exposure:

Test duration: 90 days

Frequency of treatment:

Daily
Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Low: 5 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Medium: 50 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
High: 750 mg/kg bw per day ; reduced to 500 mg/kg bw/day on day 56 due to severe effects
Basis:
actual ingested

No. of animals per sex per dose:

Male: 10 animals
Female: 10 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a range finding study
- Rationale for animal assignment (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before start, then weekly

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily inspection, no quantification

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start: control + high dose group; week 13 all treated animals


HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 90
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all survivors (day 90)
- Parameters checked: Hemoglobin, Erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin(- concentration), total leukocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, platelets, reticulocytes, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 90
- Animals fasted: No
- How many animals:all survivors (day 90)
- Parameters checked: Urea, Glucose, tot. protein, albumin, albumin/globulin ratio, sodium, potassium, calcium, chloride, inorg. phosphorus, ASAT, ALAT, Alk. phosphatase, creatinine, tot. cholesterol, tot. bilirubin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weeks 12 or 13
- Dose groups that were examined: all survivors
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, organ weights: Adrenals, brain, testes, epididymides, heart, kidneys, liver, ovaries, spleen, thymus, uterus
HISTOPATHOLOGY: Yes (see table)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortailty: high dose : 3 females on days 28, 42, 53 and after dose reduction (day 56) on days 58 and 74. Two males were killed in extremis on days 85 and 89. Clinical signs: see below

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortailty: high dose : 3 females on days 28, 42, 53 and after dose reduction (day 56) on days 58 and 74. Two males were killed in extremis on days 85 and 89. Clinical signs: see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and females of the high dose group showed reduced body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced dietary intake was evident for animals of either sex treated at the high dose throughout the study. No adverse effects were detected for animals treated with 50 or 5 mg/kg/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiencies were similarly affected as food consumption.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high dose: males & females showed microcytic hypochromic anaemia. Males had reduced clotting time, elevated mean cell hemoglobin concentration and leukocyte count.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see below

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Details on results:

Clinical observations:
Following the deaths of three high dose females, the top dose level was reduced from 750 to 500 mg/kg/day from day 56. Further deaths occurred at this dose; two high dose females were found dead on days 58 and 74 and two high dose males were killed in extremis on days 85 and 89. No mortalities occurred at other dose levels. Clinically observable signs of hunched posture were detected for 750 mg/kg/day dosed animals and deterioration in physical condition was evident, resulting in four deaths and the subsequent reduction of the dose level to 500 mg/kg/day. The reduction in dose level did not result in a regression in clinical signs and pallor of the extremities was evident for all high dose animals until th end of the study, with hunched posture observed for individual animals. Bright yellow staining was detected on cage tray-liners of high dose females. Reduced bodyweight gains were observed for high dose males throughout the study and for high dose females during the first three weeks of the study and there was reduced dietary intake for all high dose animals. No treatment-related effects were observed for animals of either sex treated with 50 or 5 mg/kg/day.

Laboratory findings:
Haematology and blood chemistry: High dose animals displayed microcytic hypochromic anaemia. High dose males had a reduction in clotting time, elevated mean cell haemoglobin concentration and leucocyte count, particularly in the lymphocyte fraction. High dose animals showed reduced albumin concentration, with reduction in total protein evident for males only. High dose animals also showed elevated alanine aminotransferase and phosphorus levels. Furthermore aspartate aminotransferase levels were elevated for males only and high dose females showed an increase in sodium concentration. No such effects were observed for animals of either sex treated with 50 or 5 mg/kg/day.

Effects in organs:
High dose males showed a reduction in absolute weight for the majority of the organs, whereas high dose females showed reduced absolute spleen, thymus, adrenal and ovary weight. High dose animals showed elevated brain, heart, kidney, and liver weight, relative to bodyweight. Adrenal and epididymis weight, relative to bodyweight was reduced for high dose males. No such effects were observed in animals treated with 50 or 5 mg/kg/day.

Treatment-related effects in organs included:

Liver: Hepatotoxicity was observed in high dose animals; morphological changes included generalised hepatocyte enlargement, single cell hepatocyte necrosis, karyomegaly and variation in nuclear size, increased mitotic activity, bile duct proliferation, eosinophilic inclusions and accumulations of pigment. These changes were seen more frequently in prematurely dead animals.

Spleen: Higher grades of severity of pigment accumulation were seen in relation to treatment in high dose males only.

Adrenals: Treatment-related cortical atrophy was seen for all males and several females in high dose groups.

Kidneys: Tubular basophilia, tubular degeneration and accumulations of eosinophilic pigment were observed in high dose animals.

Lungs: A higher incidence of accumulations of alveolar macrophages was seen in high dose animals.

Lymph Nodes: Vacuolated histiocytes were seen in the lymph nodes of high dose animals. Cervical and mesenteric nodes were affected but changes were more severe and prevalent in mesenteric nodes.

Reproductive organs: Bilateral testicular atrophy of a varying degree of severity was seen in high dose males, as was a reduction in spermatozoal content. reduced sectretory content of the prostate and seminal vesicles was also observed in high dose males. The ovaries of three high dose females contained no developing follicles.

No such effects as described above were observed for animals of either sex treated with 50 or 5 mg/kg/day.

Necropsy: Interim death animals displayed pale, red or small adrenals, dark contents present in the intestines and stomach, raised limiting ridge of the stomach, pale or mottled appearance of the liver and kidneys, and dark or pale lungs with multiple dark foci present. Pale kidneys and small darkened adrenals were detected for terminal kill high dose animals. Pale liver, gastro-intestinal tract, pituitary gland and uterus were also detected, together with small testes and seminal vesicles for individual males, and small ovaries detected for one female. No treatment-related macroscopic abnormalities were detected for animals treated with 50 or 5 mg/kg/day at terminal kill

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

other: original NCD unit is mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Clinical signs:Clinically observable signs of hunched posture was detected for 750 mg/kg/day females from Day 18, and 750 mg/kg/day males from Day 21. Deterioration in physical condition was evident for interim death females, displaying signs such as hunched posture, pilo-erection, pallor of the extremities and tiptoe gait, prior to death on Days 28, 42 and Day 53. Due to the number of deaths detected at this dose level, the high level was reduced to 500 mg/kg/day from Day 56 onwards for all high dose animals. The reduction in dose level did not result in a regression in clinical signs, and pallor of the extremities was evident for all high dose animals until the end of the study, with hunched posture also detected for individual animals.

Bright yellow staining was also detected on cage tray-liners of high dose females. Deteriorating physical condition resulted in deaths of two further females on Days 58 and 74, and two males on Days 85 and 89. No treatment-related clinical observations were detected for animals of either sex treated with 50 or 5 mg/kg/day.

Behavioural Assessment. Weekly open field arena observations confirmed the clinical signs of hunched posture, tiptoe gait and pallor of the extremities detected for high dose animals during the study. No treatment-related effects were detected for animals of either sex treated with 50 or 5 mg/kg/day.

Functional Performance Tests. No treatment-related changes were detected.

Sensory Reactivity Assessments. Elevated startle reflex responses were detected for high dose animals of either sex. No such effects were detected for animals of either sex treated with 50 or 5 mg/kg/day.

Organ Weights. High dose males showed a reduction in absolute weight for the majority of the organs, whereas high dose females showed reduced absolute spleen, thymus, adrenal and ovary weight. Animals of either sex treated at the high dose level showed elevated brain, heart, kidney and liver weight, relative to bodyweight. Adrenal and epididymis weight, relative to bodyweight

was reduced for high dose males. No such effects were detected for animals of either sex treated with 50 or 5 mg/kg/day.

Gross pathology: Interim death animals displayed pale, red or small adrenals, dark contents present in the intestines and stomach, raised limiting ridge of the stomach, pale or mottled appearance of the liver and kidneys, and dark or pale lungs with multiple dark foci present. Isolated cases of a pale pancreas/uterus, and small testesfseminal vesicles were also evident. One female was cannibalised, with most of the major organs not present at necropsy. Pale kidneys and small darkened adrenals were detected for terminal kill high dose animals of either sex. Pale liver, gastro-intestinal tract, pituitary gland and uterus were also detected, together with small testes and seminal vesicles for individual males, and small ovaries detected for one female. No treatmentrelated macroscopic abnormalities were detected for animal treated with 50 or 5 mg/kg/day at terminal kill.

Histopathology. The following treatment-related changes were observed:

LIVER: Hepatotoxicity was observed in animals of either sex treated at the high dose. Morphological changes included generalised hepatocyte enlargement, single cell hepatocyte necrosis, karyomegaly and variation in nuclear size, increased mitotic activity, bile duct

proliferation, eosinophilic inclusions and accumulations of pigment. These changes were seen more frequently in, but not restricted to, premature death animals. In addition, some animals dying or killed before scheduled termination, demonstrated periportal hepatocyte vacuolation consistent with lipid accumulation. Two premature death females were also observed to have multifocal hepatocyte necrosis and one premature death male demonstrated periportal hepatocyte degeneration/necrosis. No such effects were detected for animals of either sex treated with 50 or 5 mg/kg/day.

SPLEEN: Higher grades of severity of pigment accumulation were seen in relation to treatment for high dose males. A similar effect was not observed for high dose females, or for animals of either sex treated with 50 or 5 mg/kg/day.

HEART: Vacuolation of groups of interstitial cells was observed in the myocardium of a few animals, predominantly for premature death animals, of either sex treated with the high dose only.

PANCREAS: An oedematous appearance of the exocrine pancreas was observed in relation to treatment for a few animals, predominantly for premature death animals treated with the high dose only.

ADRENALS: Treatment-related cortical atrophy was seen for all males and several females treated with the high dose only.

KIDNEYS: Tubular basophilia, tubular degeneration and accumulations of eosinophilic pigment were observed in relation to treatment for animals of either sex treated with the high dose only.

LUNGS: A higher incidence of accumulations of alveolar macrophages was seen in relation to treatment for high dose animals of either sex. No such effects were detected at the 50 or 5 mg/kg/day dose level.

LYMPH NODES: Vacuolated histiocytes were seen as a consequence of treatment in the lymph nodes of animals of either sex treated with the high dose. Cervical and mesenteric nodes were affected but changes were more severe and more prevalent in mesenteric nodes.

THYMUS: Lymphoid atrophy was seen in relation to treatment for all high dose, premature death male or female animals for which the tissue was available.

BONE (Femur): Trabecular bone formation was generally more pronounced for male animals and possibly also for females treated with the high dose only.

BONE MARROW (Sternum): Adipose infiltration of the marrow was observed with generally higher grades of severity in male animals treated with the high dose. Such is indicative of marginal marrow hypoplasia. Female animals at this dose level were not similarly affected. No such effects were observed for animals of either sex treated with 50 or 5 mg/kg/day.

TESTES: Bilateral testicular atrophy of a varying degree of severity was seen for all male animals treated with the high dose, but not at any other dose level.

EPIDIDYMIDES: A reduction in spermatozoa1 content was observed in relation to treatment for all male animals treated with the high dose only.

PROSTATE: Reduced secretory content was seen in relation to treatment for male animals treated with the high dose only.

SEMINAL VESICLES: A treatment-related reduction in secretory content was seen for all male animals treated with the high dose only.

OVARIES: The ovaries of three females, one of which died before scheduled termination, contained no developing follicles.

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to rats, by gavage, for a period of ninety consecutive days at dose levels of up to 750 mg/kg/day (reduced to 500 mg/kg/day from Day 56) resulted in treatment-related changes at the highest dose level.
No effects were demonstrated in animals treated with 50 or 5 mg/kg/day and the "No Observed Effect Level" (NOEL) was, therefore, considered to be 50 mg/kg/day.

Executive summary:

Toxicity was characterised mainly by unspecific signs of ill health in the high dose group (reduced body weight, food consumption, clinical signs, weights of several organs)

Target organs identified by microscopy in the high dose group only were: Kidney, liver, blood, spleen, adrenals, lungs, lymph nodes, reproductive organs (testes, prostate and seminal vesicles, ovaries).

No such effects as described above were observed for animals of either sex treated with 50 or 5 mg/kg/day.