Boronic acid, (4-ethylphenyl)-

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:

2007-05-03 to 2007-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995-07-27

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996-09-30

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(4-ethylphenyl)boronic acid

EC Number:

613-145-5

Cas Number:

63139-21-9

Molecular formula:

C8 H11 B O2

IUPAC Name:

(4-ethylphenyl)boronic acid

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Füllinsdorf, Switzerland
- Age at study initiation: 7 weeks (age at delivery)
- Weight at study initiation (beginning of acclimatization):
Males: 167.1 - 171.5 g (mean 168.6 g)
Females: 137.5 - 141.1 g (mean: 139.1 g)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: Pelleted standard Provimi Kliba 3433 (batches no. 89/06, 16/07) rat maintenance diet was analyzed for contaminants
- Water: Community tap-water from Itingen was available in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples showed that none of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 28 (group 1-3), from day 0 to day 32 (group 4), from day 0 to day 42 (recovery group 1) and from day 0 to day 46 (recovery group 4)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration by gavage is a common and accepted route of exposure for studies of this type.

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Good solubility in PEG 300
- Amount of vehicle: 5 mL/kg bw
- Lot/batch No: 1300225 63006152 until 2007-05-30, then 1310049 30707244 from 2007-05-31

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability of dose formulations: At least one week based upon the results of stability analysis performed during a non-GLP 5-day dose-range-finding study.
- Storage of dose formulations: At room temperature (15 - 25 °C) in glass beakers.

ANALYSIS OF DOSE FORMULATIONS:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed according to a HPLC analytical method supplied by the sponsor and previously adapted at the test laboratory.

Duration of treatment / exposure:

- 28 days (group 1 ( 0 mg/kg bw), group 2 (25 mg/kg bw) and group 3 (100 mg/kg bw)
- 32 days (group 4 (400/300 mg/kg bw): first two days at 400 mg/kg bw, 2 days recovery, remaining 28 days at 300 mg/kg bw)
- 42 days (recovery group 1 ( 0 mg/kg bw): 28 days, then 14 days recovery period)
- 46 days (recovery group 4 (400/300 mg/kg bw): first two days at 400 mg/kg bw, 2 days recovery, remaining 28 days at 300 mg/kg bw, then 14 days recovery period)

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

after 2 days with 400 mg/kg bw/day and additional 2 days of recovery

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

for the fist two days

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups 1 (400/300 mg/kg bw) and 4 (vehicle control): 10 males and 10 females
Groups 2 (100 mg/kg bw) and 3 (25 mg/kg bw): 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of two non-GLP 5-day dose range-finding studies in which the test item was administered by gavage to 2 rats per group and sex
- Fasting period before blood sampling for clinical biochemistry: 18 hours

- Rationale for selecting satellite groups: Testing of reversibility of effects after a 2 week recovery period in the highest dose group and comparability with the vehicle group.

- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

n.a.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included: Piloerection, salivation, hunched posture, ataxia, tremor/twitching, prostration, hyperactivity, somnolence, dyspnea, tachypnea and bradypnea

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, day 8, day 15, day 22, day 28, day 32

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 wees (all dosage groups) and after 6 weeks (group 1 and group 4)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: All animals (30 males and 30 females)
- Parameters shown in table 1-3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks (all dosage groups) and after 6 weeks (group 1 and group 4)
- Animals fasted: Yes
- How many animals: All animals (30 males and 30 females)
- Parameters shown in table 4 were examined.

URINE ANALYSIS: Yes
- Time schedule for collection of urine: After 4 wees (all dosage groups) and after 6 weeks (group 1 and group 4)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters shown in table 5+6 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All animals
- Battery of functions tested: Grip strength / locomotor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7)

HISTOPATHOLOGY: Yes (see table 7): Slides of all organs and tissues listed in boldface type that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by the study pathologist. Organ and tissue samples taken from the animal which was killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group. Because possibly treatment-related morphologic changes were detected in those organs of high-dose animals, the stomach, the testes and the epididymides from animals of the mid- and low-dose group were examined.

Statistics:

STATISTICAL ANALYSIS
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory data, organ weights and ratios:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution
- Fisher's exact-test was applied to the macroscopic findings

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males (day 28 - 42 (recovery)):
- Breathing noises in 1/5 males;

400/300 mg/kg bw, males:
- All animals showed different grades of decreased activity and sedation from day 2 to day 5
- 9/10 animals showed ruffled fur on day 3 and 5/10 males showed this finding on day 4
- 5/10 animals showed prostration on day 2
- Transient salivation in 2/10 males on day 8
- Breathing noises or dyspnea in 3/10 males (day 3 and 30 to 32, day 20 to 23 and day 27 to 32, and day 2, respectively)
- A tense and enlarged stomach was palpable in 4/10 males on day 2;

100 mg/kg bw, males:
- Transient salivation in 2/5 males on day 8
- Breathing noises or dyspnea in 2/5 males on day 20 and 28 and from day 25 to 28, respectively;

400/300 mg/kg bw, females:
- Transient salivation in 1/10 females on day 8
- Transient breathing noises in 2/10 females (from day 22 and 30 to day 32, respectively)

Mortality:

mortality observed, treatment-related

Description (incidence):

One male treated with 400 mg/kg bw had to be killed in extremis after the second administration. The animal appeared highly sedated with decreased activity and prostration. This death was considered to be test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males:
- Weight loss up to 4.5% in 6/9 males on day 8 (statistically significant)
- Recovery after reduction of the daily dose;

400/300 mg/kg bw, males (day 28-42 (recovery)):
- Significant weight gain in males on day 36 and 42;

400/300 mg/kg bw, females:
- Significant weight loss in 1/10 females on day 8;

400/300 mg/kg bw, females (day 28-42 (recovery)):
- Significant weight gain in females on day 28, 36 and 42

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Relative food consumption in test item-treated males at all dose levels was slightly lower than in controls throughout treatment. The decrease in food consumption of test item-treated males is considered to be test item related. Absolute and relative (except for those treated with 25 mg/kg bw in week 3) food consumption in test item treated females at all dose levels was slightly higher than in controls with the difference reaching statistical significance in absolute food consumption in week 4 in those treated with 100 or 400/300 mg/kg bw. Theses effects are considered not to be test item related.

Food efficiency:

not examined

Description (incidence and severity):

not examined

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

not examined

Description (incidence and severity):

not examined

Ophthalmological findings:

not examined

Description (incidence and severity):

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males (day 42):
- Decrease in hemoglobin concentration distribution width, (significant but within historical range)
- Decrease in red cell volume distribution (significant but within historical range)

400/300 mg/kg bw, males (day 28):
- Slight but significant decrease of hematocrit
- Increase in reticulocyte count (significant but no dose-response relationship)
- Slight shift towards younger reticulocytes in reticulocyte maturity index (significant but no dose-response relationship)
- Decrease in white blood cell count (significant but no dose-response relationship);

100 mg/kg bw, males (day 28):
- Methemoglobin decrease (out of the range of historical data, but no dose-response relationship);

400/300 mg/kg bw, females (day 42):
- Decrease in hemoglobin concentration distribution width (significant but within historical range)
- Decrease in red cell volume distribution (significant but within historical range);


400/300 mg/kg bw, females (day 28):
- Slight decrease of hematocrit
- Decrease in white blood cell count (not significant, no dose-relation)
- Methemoglobin increase (significant, no dose-relation);

100 mg/kg bw, females (day 28)
- Decrease in white blood cell count (not significant, no dose-relation);

- 25 mg/kg bw, females (day 28):
- Decrease in white blood cell count (not significant, no dose-relation)
- Mean cell hemoglobin and mean cell hemoglobin concentration were decreased (significant but without dose relation)
- Methemoglobin decrease (out of the range of historical data, but no dose-response relationship);

Several statistically significant changes were noted in absolute and relative differential white blood cell count. These changes were inconsistent across sexes, without dose-relation and within the normal range of historical data for rats of this strain and age.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following and all other changes noted in clinical chemistry were without dose-relation, inconsistent across sexes, and/or within the normal range of historical data for rats of this strain and age. Thus, they were considered not to be test item-related. Statistical significance was attained as indicated:

400/300 mg/kg bw, males (day 42):
- Decreased glucose levels (significant)
- Increased urea
- Increased triglycerides;

400/300 mg/kg bw, males (day 28):
- Slightly increased urea
- Increased cholesterol
- Decreased triglycerides
- Increased sodium
- Increased phosphorus (significant)
- Decrease in total protein (significant)
- Decreased globulin (significant)
- Increased alanine-aminotranferase (significant);

100 mg/kg bw, males (day 28):
- Decrease in glucose (significant)
- Decreased cholesterol
- Decreased triglycerides
- Increased phosphorus;

25 mg/kg bw, males (day 28):
- Decreased triglycerides;

300/400 mg/kg bw, females (day 42):
- Decreased glucose levels (significant)
- Increased triglycerides
- Increased aspartate-aminotranferase (significant);

300/400 mg/kg bw, females (day 28):
- Slightly increased urea
- Decreased cholesterol
- Increased triglycerides
- Decreased phosphorus
- Decrease in total protein (significant)
- Decreased globulin (significant);

100 mg/kg bw, females (day 28):
- Slightly increased urea (significant)
- Decreased cholesterol
- Decreased triglycerides
- Increased globulin (significant);

25 mg/kg bw, females (day 28):
- Slightly increased urea
- Decreased cholesterol
- Decreased triglycerides
- Increased alanine-aminotranferase (significant)

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males (day 28):
- Slight increase in urine pH (statistically significant but within the normal range of historical data);

400/300 mg/kg bw, females (day 28):
- Increased urine volume (statistically significant but within the normal range of historical data)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects observed.

Immunological findings:

not examined

Description (incidence and severity):

Not examined.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males (day 42)
- Decrease in absolute and relative liver weights;

400/300 mg/kg bw, males (day 28)
- Slight increase in absolute and relative liver weights (statistically significant)
- Increased absolute and relative kidney weights (significant increase in kidney to brain weight ratio);

25 mg/kg bw, males (day 28)
- Slight increase in absolute and relative liver weights;

400/300 mg/kg bw, females (day 42)
- Slight increase in absolute and relative liver weights;

400/300 mg/kg bw, females (day 28)
- Increased in absolute and relative kidney weights (statistically significant);

All doses, females (day 28)
- Increase in absolute liver weights (dose-response relationship, but no statistical significance)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related findings:
400/300 mg/kg bw, males (day 28):
- Distended stomach (1 male which had to be killed in extremis on day 2) and a tense and enlarged stomach (other 4 males);

Non test item-related findings:
400/300 mg/kg bw, males (day 28)
- Foci in the thymus (2 animals)
- Foci in a mandibular lymph node (1 animal);

400/300 mg/kg bw, females (day 28)
- Foci in the thymus (1 animal);

400/300 mg/kg bw, females (day 42)
- One nodule in the uterine adipose tissue (1 animal);

These findings were considered to be within the range of normal background lesions, which may be seen in species of this strain and age in this study type and were considered incidental, reflecting the usual individual variability.

Neuropathological findings:

not examined

Description (incidence and severity):

Not examined.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

400/300 mg/kg bw, males (day 42)
- Minimal tubular degeneration (1 animal);

400/300 mg/kg bw, males (day 28)
- Minimal focal erosion and parakeratosis (all animals)
- Minimal and slight tubular degeneration (bilaterally) in the testes (2 animals) with minimal cellular detritus in the epididymides (1 animal)
- Moderate acute inflammation of the forestomach, moderate submucosal edema mainly in the glandular stomach, slight increase of lymphoid apoptosis in the mesenteric lymph node, minimal degrees of tubular degeneration in the testes as well as of cellular detritus in the epididymides (one male which had to be killed in extremis on day 2);

400/300 mg/kg bw, females (day 28)
- Minimal focal erosion and parakeratosis (all animals);

The incidence and severity recorded are within the range of normal background
alterations that may be recorded in animals of this strange and age.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No effects observed.

Other effects:

no effects observed

Description (incidence and severity):

No other effects observed.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related adverse effects were observed.

2

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test substance related effects were observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, 100 mg/kg body weight/day of the test item were established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the test item as the no-observed-adverse-effect-level (NOAEL). Treatment with 400 mg/kg/day for two days was not considered to be tolerated by the rats because of the clinical signs and the premature sacrifice (in extremis) of one male.

Executive summary:

GENERAL

In this subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 25, 100 and 400/300 mg/kg body weight/day for a period of 28 days. The high dose group received 400 mg/kg/day for two days. Due to severe signs of toxicity, administration was suspended for two days in this group and re-started with the lower dose of 300 mg/kg/day on day 5. Treatment with 300 mg/kg/day was continued for 28 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 400/300 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. Because of possible test item-related findings noted in the high dose group, the stomach, the testes and the epididymides were also examined in the animals of groups 2 and 3.

 

DOSE FORMULATION ANALYSIS

The results obtained in dose formulation analysis confirmed the correct preparation and storage of application formulations during the conduct of this study.

 

MORTALITY / VIABILITY

One male treated with 400 mg/kg/day had to be killed in extremis after the second administration. This death was considered to be test item-related.

 

CLINICAL SIGNS

All males treated with 400 mg/kg/day showed different grades of decreased activity and sedation from day 2 to day 5 at the latest. Nine of these males additionally showed ruffled fur on day 3 and five males showed this finding on day 4. Five males treated with 400 mg/kg/day showed prostration on day 2. A tense and enlarged stomach was palpable in four males treated with 400 mg/kg/day on day 2. These findings were clearly test item-related. The male treated with 400 mg/kg/day which had to be killed in extremis appeared highly sedated with decreased activity and prostration. Due to these severe signs of toxicity in males treated with 400 mg/kg/day after the second administration, administration of animals treated with 400 mg/kg/day was suspended for two days and re-started with a lower dose of 300 mg/kg/day on day 5. During treatment with 300 mg/kg/day, transient salivation was noted in two males each treated with 100 or 400/300 mg/kg/day and in one female treated with 400/300 mg/kg/day. Two females treated with 400/300 mg/kg/day transiently showed breathing noises. Breathing noises or dyspnea were also noted in two males treated with 100 mg/kg/day, as well as in 3 males treated with 400/300 mg/kg/day. Salivation and breathing noises are considered to be treatment and possibly test item-related. During recovery, no clinical signs were noted in females at any dose level or males treated with 25 or 100 mg/kg/day. In one male (no. 29) treated with 400/300 mg/kg/day, breathing noises were still present throughout recovery.

 

DETAILED CLINICAL OBSERVATIONS (WEEKLY)

No clinical signs were evident during weekly detailed observations (weeks 1 to 3).

 

FUNCTIONAL OBSERVATIONAL BATTERY

No differences in the functional observational battery were noted at week 4 of treatment.

Grip Strength: No differences were noted in the mean grip strength when compared with the controls.

Locomotor Activity: No test item-related differences were noted in the mean locomotor activity when compared with the controls.

 

FOOD CONSUMPTION

Contrary effects noted in absolute food consumption of test item treated females at all dose levels and males treated with 100 mg/kg/day were considered not to be test item-related. In males treated with 400/300 mg/kg/day, absolute food consumption was moderately lower than in controls in week 1 of treatment but improved in the following weeks. Relative food consumption in test item-treated males at all dose levels was slightly lower than in controls throughout treatment. During recovery, absolute and relative food consumption of males treated with 400/300 mg/kg/day was lower than that of the respective controls. In females treated with 400/300 mg/kg/day, absolute food consumption was slightly higher and relative food consumption - in week 2 of recovery - slightly lower than that of the respective controls. The decrease in food consumption of test item-treated males is considered to be test item related.

 

BODY WEIGHT

In males treated with 400/300 mg/kg/day, mean body weight was lower than in controls on days 8 and 15 with the difference reaching statistical significance on day 8. A slight but clear weight loss (up to 4.5%) was noted on day 8 in six of nine males and one of ten females treated with 400/300 mg/kg/day. Consequently, the mean body weight of these males on day 8 was 8% lower than that of the respective controls and the mean body weight loss was statistically significant compared to controls. After reduction of the daily dose, both parameters showed a clear recovery and the mean body weight gain of males treated with 400/300 mg/kg/day on day 28 was higher than in all other groups. The weight loss is considered to be test item-related. During recovery, mean body weight of males and females treated with 400/300 mg/kg/day was higher than that of controls with the difference being statistically significant on days 8 and 14 in males and additionally on day 1 in females. Furthermore, mean body weight gain of males treated with 400/300 mg/kg/day was higher than that of the respective controls (statistically significant on day 8 of recovery).

 

CLINICAL LABORATORY INVESTIGATIONS

Hematology: No test item-related findings were noted in hematology parameters.

Clinical Biochemistry: All changes noted in clinical chemistry, even if statistically significant, were without doserelation, inconsistent across sexes, and/or within the normal range of historical data for rats of this strain and age. Thus, they were considered not to be test item-related.

Urine alysis: No test item-related changes were noted in urinalysis parameters.

 

ORGAN WEIGHTS

Slightly increased absolute and relative liver weights were noted in males treated with 25 or 400/300 mg/kg/day with the difference reaching statistical significance at 400/300 mg/kg/day in absolute weight and liver to brain weight ratio. In females, mean liver weights were increased with relation to dose but without statistical significance. After the recovery period, absolute and relative liver weights were decreased in males treated with 400/300 mg/kg/day In females treated with 400/300 mg/kg/day, absolute liver weight and liver to brain weight ratio were slightly increased. Increased absolute and relative kidney weights were noted in males and females treated with 400/300 mg/kg/day with the difference reaching statistical significance in females in absolute weight and in males and females in kidney to brain weight ratio. Absolute and relative kidney weights were still slightly increased in males and females treated with 400/300 mg/kg/day except kidney to body weight ratio in females. Changes in liver weights were of small magnitude and reversed during recovery. Thus, they are considered adaptive in nature. Changes in kidney weights are, in the absence of corresponding microscopic findings, considered not to be test item-related.

 

MACROSCOPIC / MICROSCOPIC FINDINGS

The male treated with 400 mg/kg/day, which had to be killed in extremis on day 2 had a distended stomach. All other macroscopic findings were within the range of normal background lesions, which may be seen in species of this strain and age in this study type and were considered incidental, reflecting usual individual variability. Microscopically, in the animal killed in extremis, changes consisting of moderate acute inflammation of the forestomach, which correlated macroscopically with distension of the forestomach, and of moderate submucosal edema in the glandular stomach were noted. Further, a slight increase of lymphoid apoptosis was recorded in the mesenteric lymph node of this animal. These findings were considered to be of adverse nature. Minimal degrees of focal erosion and parakeratosis were recorded in the forestomach of main study males and females treated with 400/300 mg/kg/day. After the recovery period, these lesions showed complete regression. These findings were considered to be test item-related but non-adverse.

 

ASSESSMENT

Oral administration of the test item to Wistar rats at doses of 25, 100 and 400/300 mg/kg/day, for 28 days resulted in no effects on grip strength or locomotor activity, hematology parameters, clinical chemistry or urinalysis. One male had to be killed in extremis after treatment with 400 mg/kg/day for two days. This male had a distended stomach with moderate acute inflammation of the forestomach and moderate submucosal edema in the glandular stomach. A slight increase of lymphoid apoptosis was recorded in the mesenteric lymph node of this animal. Test item-related findings were decreased activity, sedation, prostration, ruffled fur and an enlarged and tense palpable stomach, reduced food consumption, and weight loss in males during and some days (week 1) after treatment with 400 mg/kg/day. After suspension of administration for two days and consequent reduction of the daily dose to 300 mg/kg/day, these parameters showed a clear recovery. Transient salivation or breathing noises or dyspnea were noted in single males treated with 100 mg/kg/day and in single animals of both sexes of the high dose group during treatment with 300 mg/kg/day. In one male treated with 400/300 mg/kg/day, breathing noises were still present throughout recovery. These clinical signs are considered to be treatment and possibly test item-related but non adverse. Increases in liver weights in animals treated with 400/300 mg/kg/day were of small magnitude and reversed during recovery. They are considered to be adaptive in nature. Increases in kidney weights at the same dose level are, in the absence of corresponding microscopic findings, considered not to be test item-related. Microscopically, minimal degrees of focal erosion and parakeratosis were recorded in the forestomach of males and females treated with 400/300 mg/kg/day. After the recovery period, these lesions showed complete regression. These findings were considered to be test itemrelated but non-adverse. Based on the results of this study, 100 mg/kg body weight/day of the test item were established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the test item as the no-observed-adverse-effect-level (NOAEL). Treatment with 400 mg/kg/day for two days was not considered to be tolerated by the rats because of the clinical signs and the premature sacrifice (in extremis) of one male.