Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 1996 - 22 August 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
MMBC
Appearance: tan solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Kingston, Stone Ridge, NY
- Age at study initiation: Approximately 6 weeks
- Housing: The animals were housed individually in suspended stainless steel cages (7 x13.5 x7 in., i.e. 18 x 34 x 18 cm) with wire mesh fronts and bottoms. Clean cage banks were supplied at approximately 2 week intervals. Cages were suspended above absorbent-paper pan liners which were changed 3 times a week.
- Diet (e.g. ad libitum): PMI Certified Rodent Diet 5002(M) (Purina Mills Inc., Richmond, IN).
- Water (e.g. ad libitum): ad libitum access to filtered tap water (via automatic watering).
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24 °C
- Humidity (%): 40 - 50 %
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness

IN-LIFE DATES: From: To: 24 July 1996 - 22 August 1996

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSE PREPARATION AND ADMINISTRATION
Suspensions of the test material in corn oil were prepared daily for administration (at 5 mL/kg of body weight) at dose levels of 15, 150 or 750 mg a.i./kg. An aliquot of the test substance and the corn oil vehicle was heated in an oven set to maintain approximately 45°C (a separate aliquot was used for each day of dose preparation to avoid reheating the same sample and potential chemical breakdown). The appropriate amount of test substance was weighed into a tripour disposable beaker for each dose level. The appropriate volume was reached by adding the heated corn oil. The material was suspended via a magnetic stir bar/stir plate. The suspensions were placed back in the oven until just prior to dosing. The suspensions were allowed to stir prior to dosing to ensure a homogeneous mixture.

Stability of test substance in corn oil for 4 hrs was confirmed by analysis of 3 and 200 mg a.i./mL prior to the initiation of dosing. Homogeneity (top, middle and bottom samples on day 0) and proximity to target concentration samples (day 0, 14 and 28) were sent for analytical determinations. All samples were collected at the end of the dosing period. All dose suspensions were stirred continuously throughout dose administration and sampling. Doses were calculated on the animal's most recent weekly body weight. All animals were dosed within 4 hrs of dose preparation.

The control group was given the vehicle only, using the same route and volume as the treated groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYTICAL PROCEDURES
Recovery of test substance from dosing suspensions was determined from analysis of fortified samples. Control corn oil samples were used for the fortification of test substance at different levels.
Two mL control samples of corn oil were pipetted into 100 mL graduated cylinders and fortified with test substance at 60,000 µg, 300,000 µg, and 400,000 µg by weighing solid Technical standard into the cylinder, and at 200 µg (LOQ recovery) and 6000 µg by using standard prepared in acetone. Samples were extracted/diluted with acetonitrile and HPLC mobile phase (acetonitrile:water 50:50 v:v). Extracts were analyzed by HPLC with UV spectrophotometer. Test samples at 2.0 mL were extracted and analyzed in the same manner as fortified samples.

HPLC Conditions

Instrument: Shimadzu LC-6A
Detector: Shimadzu SPD-6A uv Spectrophotometer
Column: Supelcosil LC 8 DB ID 4.6 mm x 25 cm
Mobile Phase: acetonitrile:water (50:50 v:v)
Temperature: 35°C
Flow Rate: 1.0 mL/minute
Wave Length: 204 nm
Absorbance: 0.32
Injection Volume: 30 µL


DATA COLLECTION AND CALCULATION
Data was collected using a Hewlett Packard work station with Nelson System Inc. software (Nelson System). The Nelson System was used to prepare the appropriate sequence and method files for the analysis of the samples and the collection of the data. The concentration of test substance was calculated using the Nelson System, by plotting a curve (linear constrained) of various standard concentrations against their peak heights, determining standard curve coefficients by least-squares analysis, and calculating values for unknown samples using these coefficients.

METHOD PERFORMANCE
Sample mg/mL levels in the run were adjusted for the average daily recovery. The average of all 10 non-LOQ recoveries was 95.5 % with cv = 3.6 %, and ranged from 91.0 - 102 %, which was within an acceptable range of 70 - 120 %. The LOQ recovery run at 200 µg was 92.5 %.

HOMOGENEITY

Analysis of top, middle, and bottom fractions for dosing suspension samples prepared at the beginning of the study showed average percents of target of 98.7 % (cv = 0.66 %) for 3.0 mg/mL samples, 102 % (cv = 0.57 %) for 30.0 mg/mL samples, and 103 % (cv = 0.56 %) for 150 mg/mL samples. All samples were within 3 % of target concentration with cv less than 1 %, indicating that the samples were homogeneous.

STORAGE STABILITY
Analysis of stability samples obtained from the 3 mg/mL and 200 mg/mL dosing suspensions immediately after preparation and approximately 4 hours after preparation indicated that the suspensions were at 94 % (for 3 mg/mL) and 102 % (for 200 mg/mL) of nominal concentration. These results demonstrate that the dosing suspensions were stable for approximately 4 hours.

PROXIMITY TO TARGET
Analyses of samples for proximity to target were within ± 3 % of nominal concentrations. These results indicate that dosing suspensions were adequately prepared and that nominal concentrations were an accurate reflection of levels administered to the test animals.

CONCLUSION
Based on the results of the above experiment it is concluded that the dosing suspensions were within acceptable proximity to target concentrations each day of sampling and over each dosing level, that samples were homogeneous over top, middle, and bottom sampling areas, and that concentrations were stable for at least 4 hours.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
15 mg ai./kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg a.i./kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg a.i./kg
Basis:
actual ingested
No. of animals per sex per dose:
6 animals per sex per dose.
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
A careful cageside examination was made at least once each day. Each rat was observed for signs of ill health or reaction to treatment. On each treatment day animals were observed in the afternoon for mortality only.

Body weights and feed consumption were determined weekly for all animals beginning 1 week prior to the start of dosing. A terminal body weight was determined for each animal immediately prior to necropsy and after an overnight fast.

Haematology and clinical chemistry measurements were performed on all animals at the terminal necropsy. Animals were fasted overnight and blood samples were collected just prior to the terminal necropsy. All samples were collected from the abdominal aorta of rats that had been anesthetized with sodium pentobarbital (Nembutal, 50 mg/mL, Abbott Laboratories, North Chicago, IL) administered intraperitoneally at approximately 0.1 mL/100 g body weight. Blood samples were collected in an order that rotated through treatment groups (i.e., one animal from each treatment group was bled before a second animal from the same group). Once blood was collected, animals were exsanguinated via the abdominal aorta.

The haematology and clinical chemistry parameters that were evaluated are listed below.

Haematology
Haematocrit (HCT)
Erythrocytes (RBC)
Haemoglobin (HGB)
Mean Cell Volume (MCV)
Total White Blood Cell Count (WBC) and Differential (WBC DIFF)
Platelets (PLT)
Mean Cell Haemoglobin (MCH)
Mean Cell Haemoglobin Concentration (MCHC)

Clinical Chemistry
Serum Glutamic Pyruvic Transaminase (GPT) (also known as serum alanine aminotransferase)
Serum Glutamic Oxaloacetic Transaminase (GOT) (also known as serum aspartate aminotransferase)
Cholesterol (CHOL)
Triglycerides (TRIG)
Total Bilirubin (BILI)
Alkaline Phosphatase (ALP)
Glucose (GLU)
Gamma Glutamyl Transpeptidase (GGT)
Blood Urea Nitrogen (BUN)
Creatinine (CREA)
Total Protein (TP)
Albumin (ALB)
Globulin (GLOB)
Albumin/globulin ratio (A/G)
Calcium (CA)
Chloride (CL)
Sodium (NA)
Potassium (K)
Inorganic Phosphorus (PHOS)

All haematology parameters excluding white blood cell differential counts were determined using a Baker 9000 Haematology Analyzer (Serono-Baker Corp., Allentown, PA). White blood cell differential counts were determined by microscopic examination. All clinical chemistry parameters were analyzed using a Hitachi 704 Random-Access Chemistry System (Boehringer Mannheim Diagnostics, Indianapolis, IN).
Sacrifice and pathology:
At the end of the 28-day treatment period, all surviving rats were anesthetized with an intraperitoneal injection of sodium pentobarbital, euthanized by exsanguination and necropsied. All organs, tissues and body cavities of the euthanized animals were examined and gross abnormalities were recorded. The following organs were weighed from each rat:
adrenals (2)
liver
kidneys (2)
testes (2)

Relative organ weights were calculated as a percentage of terminal body weight. The following tissues from all rats were preserved in 10 % buffered formalin:

adrenals (2) pituitary
brain prostate
cecum rectum
colon salivary gland
duodenum sciatic nerve
esophagus seminal vesicles
femur with joint gross lesions (to include a border of apparently normal tissue) skeletal muscle
heart skin
ileum spleen
jejunum stomach
kidneys (2) testes (2) (including epididymus)
larynx thymus (where present)
liver (2 lobes, median and left lateral) thyroid/ parathyroid
lungs tongue
lymph nodes (cervical and mesenteric) trachea
mammary glands urinary bladder
ovaries uterus (with cervix)
pancreas vagina

Microscopic examinations were performed on the adrenals and testes from animals in the 0 mg/kg (Control) and 750 mg/kg groups and on the stomachs, livers, kidneys and all gross lesions from animals in all groups.

MATERIALS AND METHODS
All animals were given a complete gross necropsy. Tissues listed in above were collected from all animals and saved in 10 % neutral buffered formalin. The stomachs were inflated with formalin at necropsy, and were opened and inspected for gross lesions post-fixation. Adrenals and testes from the high dose and control animals, as well as the stomachs, livers, kidneys and all gross lesions from all animals were embedded in paraffin, sectioned at approximately 5 to 6 microns and stained with haematoxylin and eosin.

The severity of microscopic changes was graded on a scale of 1 - 5. The grading scheme for some of the treatment-related gastric changes is defined in greater detail in the results section. The scale for other gradeable findings is as follows:

1 = Minimal - The least amount or degree of change detectable, affecting approximately 5 % or less of the tissue.

2 = Slight - A change that is easily detected, but of limited degree, amount, or intensity, affecting approximately 6 - 25 % of the tissue. Other adjectives that may be used to indicate a grade of +2 would include, mild, small, few, etc.

3 = Moderate - This amount or degree of change is clearly visible, and usually relatively prominent and extensive. This grade indicates that large or multiple areas are involved. This grade would be appropriate when a change affects approximately 26 - 60 % of a tissue.

4 = Moderately Severe - This category is used when the change involves the majority (approx. 61 - 80 %), of the tissue examined.

5 = Severe - This category indicates the change is approaching the most severe or extensive level theoretically possible, with essentially all areas (>80 %) of the tissue involved to a very significant degree. This degree would certainly be at a level which could greatly compromise the animal, and may or may not be reversible.

Microscopic changes of the type that could not be accurately graded according to severity are simply identified as present or absent.

Histopathological examination of hearts and spleens using a light microscope from two dose groups was carried out. The examinations were performed in the control group (dosed with corn oil) and high dose group 4 (dosed with 750 mg a.i./kg/day).
Statistics:
Distributions of the following parameters were inspected for normality and homogeneity of variance across treatment groups, sexes and sampling times: body weight, feed consumption, clinical chemistries, haematology, and organ weights. Analysis of variance models were used to assess the presence or absence of an overall treatment effect. When a significant overall treatment effect was found, comparisons between control and appropriate compound-exposed groups were made using Dunnett's t-test. Statistical significance was indicated when a p-value of 0.05 or less (Dunnett's criterion) was obtained.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no mortalities or clinical signs indicative of systemic toxicity in any of the treatment groups.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities or clinical signs indicative of systemic toxicity in any of the treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg, there was a slight decrease in mean body weight and cumulative body weight change among males that progressed through the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related effects on feed consumption were noted in either sex in any of the treatment groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related hematological effects were noted.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See below for details.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below for details.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See below for details.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See below for details.
Details on results:
Mortality and Clinical Signs
There were no mortalities or clinical signs indicative of systemic toxicity in any of the treatment groups. Red stain surrounding the eyes was observed periodically in a few animals. However, this finding was considered incidental and not related to treatment since it was transient and did not follow a dose-response relationship.

Body Weight (Tables 1 and 2)
At 750 mg/kg, there was a slight decrease in mean body weight and cumulative body weight change among males that progressed through the treatment period. There were no treatment-related body weight effects in females in any of the treatment groups.
An increase in female body weight and cumulative body weight change (statistically significant on weeks 3 and 4 at 150 and 750 mg/kg) was noted in all MMBC treated groups relative to control during the treatment period. These increases have no toxicological significance due to the direction of the changes.

Feed Consumption
No treatment-related effects on feed consumption were noted in either sex in any of the treatment groups. An increase in feed consumption among females (statistically significant on weeks 2, 3, and 4 at 150 and 750 mg/kg) was noted in all groups during the treatment period. These increases have no toxicological significance due to the direction of the changes.

Haematology
No treatment-related hematological effects were noted. A statistically significant decrease in RBC was observed among females at 150 mg/kg. This finding was considered spurious since similar findings were not seen at a higher dose level.

Clinical Chemistry (Table 3)
No treatment-related changes in clinical chemistry parameters were noted at doses up to and including 150 mg/kg. A treatment-related increase in alkaline phosphatase (47%) was noted in males at 750 mg/kg. A treatment-related decrease in total protein (11%) and globulin (41%) with a corresponding increase in albumin-globulin ratio (83%) was noted in males at 750 mg/kg. However, none of these findings were evident in females. These changes may be related to the irritation type changes seen in the stomach of the test animals at 750 mg/kg. The toxicological significance of the clinical chemistry changes is not clear since they were only seen in one sex and the gastric changes were seen equally in both sexes.

Other statistically significant changes in clinical chemistry parameters were noted, but were considered incidental and not related to treatment. Increased BUN in males and decreased BUN in females at 150 and 750 mg/kg were considered incidental since the magnitude of the changes was small and the changes were in opposite directions in the two sexes. Increased glucose levels in males at 150 and 750 mg/kg were minimal and only seen in one sex and may be related to the stress of gastric irritation or fasting or a combination of the two, but not judged an indication of test substance toxicity. Calcium levels were decreased in males at 750 mg/kg, but the magnitude of the change was minimal and was only seen in one sex.

Organ Weights (Table 4)
No treatment-related changes in organ weights were noted at 15 mg/kg. Treatment-related increases in absolute (18 - 52 %) and relative (11 - 47 %) liver weights were noted at 150 and 750 mg/kg in both sexes. The liver weight changes at 750 mg/kg were accompanied by histopathologic findings in both sexes. The liver weight changes at 150 mg/kg were minimal (11 - 24 %) and were not accompanied by any histopathologic findings. In males, both the absolute and relative liver weights were increased by 18 %, but only the relative liver weight was statistically significant. Mean terminal body weights of the control and 150 mg/kg males were virtually identical. In females at 150 mg/kg, absolute liver weights were increased by 24 % and relative by 11 %. Terminal body weights were also increased by 12 % in the 150 mg/kg females and this was considered a contributory factor. The liver weight increases at 150 mg/kg in both sexes are considered treatment-related. Since these changes were minimal and had no histopathological correlate, the toxicological significance of this finding is unclear and these changes may not represent an adverse effect.

Other statistically significant organ weight changes were noted, but were judged incidental and not treatment-related. A statistically significant increase in kidney weights at 150 mg/kg (absolute) and 750 mg/kg (absolute and relative) was seen in females; however, this effect was considered secondary to an increased terminal body weight in these groups. Similar effects were not evident in males at any dose. A statistically significant increase in absolute adrenal weight was observed at 15 mg/kg in males. However, this finding was considered spurious since similar findings were not evident at higher dose levels.

Gross Pathology
No treatment-related gross pathological findings were observed at 15 or 150 mg/kg. The following treatment-related gross pathologic findings were evident at 750 mg/kg: thickened mucosa or raised focus/area in the forestomach among males and females; and, thickening of the glandular mucosa was evident in one male. All other findings were considered incidental.

Histopathology
No treatment-related microscopic changes were observed at 15 or 150 mg/kg. The following treatment-related microscopic findings were evident in males and/or females at 750 mg/kg: hyperplasia, hyperkeratosis, oedema, inflammation and ulceration/erosion of the forestomach; submucosal oedema of the glandular stomach; and, diffuse hepatocellular hypertrophy in the liver.
Examination of the hearts and spleens from animals in groups 1 and 4 showed no effects related to test article administration in male or female rats.

Effect levels

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant findings observed at this dose level other than a minimal increase in liver weight without a histopathological correlate.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Mean Body weight Values (g)

Males (6 animals per group)

 

Group

Dose

(mg/kg)

 

Observation Periods (weeks)

Pretest

Dosing Phase

0

0

1

2

3

4

1

0

Mean

SD

165.2

9.4

229.4

10.3

285.1

16.9

330.8

27.7

367.0

35.9

399.5

39.0

2

15

Mean

SD

162.9

6.8

226.6

11.1

284.1

14.6

335.7

21.0

378.2

28.3

413.4

33.4

3

150

Mean

SD

163.7

6.9

228.5

9.9

279.3

18.8

326.9

20.7

367.7

20.9

401.5

22.1

4

750

Mean

SD

163.7

9.2

229.6

9.8

273.7

11.8

313.3

19.4

345.9

26.9

359.1

35.7

 

Table 2 Mean Cumulative Change Body Weight (g)

Males (6 animals per group)

 

Group

Dose

(mg/kg)

 

Observation Periods (weeks)

Dosing Phase

1

2

3

4

1

0

Mean

SD

55.7

10.6

101.4

22.6

137.7

30.6

170.2

34.4

2

15

Mean

SD

57.6

6.7

109.2

12.4

151.6

19.3

186.9

24.6

3

150

Mean

SD

50.8

13.4

98.4

15.1

139.2

13.6

173.0

12.8

4

750

Mean

SD

44.2

4.4

83.7

11.5

116.4

19.7

129.6

29.5

 

Table 3 Mean Clinical Chemistry Values

Males (6 animals per group)

Group

Dose

(mg/kg)

 

GOT

U/L

GPT

U/L

ALP

U/L

GGT

U/L

CA

mg/DL

PHOS

mg/DL

TRIG

mg/DL

ALB

g/DL

TP

g/DL

BUN

mg/DL

1

 

0

Mean

SD

111

26

28

4

324

32

0

0

9.8

0.2

9.2

0.8

69

16

4.0

0.2

5.6

0.3

10.2

1.2

2

 

15

Mean

SD

112

41

26

6

348

119

0

0

9.8

0.2

9.0

0.7

74

21

4.0

0.2

5.6

0.2

11.6

1.5

3

150

Mean

SD

96

29

28

6

407

71

0

0

9.8

0.2

9.1

0.8

59

27

4.1

0.2

5.6

0.2

12.7*

2.1

4

 

750

Mean

SD

111

35

36

7

476*

96

0

0

9.3*

0.2

8.6

0.6

47

14

4.1

0.2

5.0*

0.3

13.0*

1.4

GOT=Glutamic Oxaloacetic Transaminase                     GPT=Glutamic Pyruvic Transaminase              

ALP=Alkaline Phosphatase                                       CA=Calcium                                                                          

PHOS=Inorganic Phosphorus                                     TRIG=Triglyceride               

BUN=Blood Urea Nitrogen                                        ALB=Albumin                                       

TP =Total Protein                                                   GGT=Gamma-Glutamyl Transferase 

mg/DL=Milligram/Decilitre                                         U/L=Units/Litre                   

g/DL=Grams/Decilitre                                               SD=Standard Deviation   

* Indicates a Statistically Significant Difference from control (p<0.05).

Only 5 values measured.

Males (6 animals per group)

Group

Dose

(mg/kg)

 

CREA

mg/DL

BILI

mg/DL

GLU

mg/DL

CHOL

mg/DL

NA

mmol/L

K

mmol/L

CL

mmol/L

AG

Ratio

GLOB

g/DL

1

 

0

Mean

SD

0.6

0.1

0.1

0.0

109

13

51

8

146

1

4.4

0.4

106

3

2.4

0.2

1.7

0.1

2

 

15

Mean

SD

0.6

0.0

0.2

0.2

120

19

52

15

146

2

4.7

0.8

106

2

2.5

0.2

1.6

0.1

3

150

Mean

SD

0.6

0.0

0.1

0.1

137*

8

51

11

146

1

4.5

0.2

105

2

2.7

0.2

1.5

0.1

4

 

750

Mean

SD

0.6

0.0

0.1

0.0

170*

14

44

12

144

2

4.5

0.3

104

3

4.4*

0.5

1.0*

0.1

CREA=Creatinine                  BILI=Total Bilirubin                               

GLU=Glucose        CHOL=Cholesterol                              

NA=Sodium                          K=Potassium        

CL=Chloride                           GLOB=Globulin                    

AG=Albumin Globulin Ratio

mg/DL=Milligram/Decilitre         mmol/L=Millimole/Litre                    

g/DL=Gram/Decilitre SD=Standard Deviation      

* Indicates a Statistically Significant Difference from Control (p<0.05).

Only 5 values measured.

 

Table 4 Absolute and Relative Organ Weights (Mean Values)

Males (6 animals per group)

Group

Dosage (mg/kg)

 

Terminal Body Weight (g)

Absolute Adrenal (g)

Relative Adrenal (%)

Absolute Testis (g)

Relative Testis (%)

Absolute Kidney (g)

Relative Kidney (%)

Absolute Liver (g)

Relative Liver (%)

1

0

Mean

SD

376.2

39..0

0.054

0.007

0.014

0.002

3.162

0.102

0.848

0.094

2.989

0.325

0.797

0.070

11.840

1.785

3.137

0.200

2

15

Mean

SD

389.5

33.9

0.063*

0.008

0.016

0.003

2.996

0.539

0.781

0.186

3.010

0.235

0.774

0.030

12.375

1.056

3.185

0.231

3

150

Mean

SD

376.3

22.3

0.056

0.004

0.015

0.001

3.260

0.105

0.869

0.060

3.704

1.407

0.980

0.352

13.967

1.479

3.704*

0.179

4

750

Mean

SD

332.4

32.0

0.055

0.003

0.017

0.002

3.142

0.571

0.943

0.130

3.227

0.368

0.970

0.047

15.331*

1.825

4.608

0.235

Relative organ weight = (absolute organ weight x 100) / terminal body weight (%)

*Indicates a statistically significant difference from the control group (p<0.05)

 

Females (6 animals per group)

Group

Dosage (mg/kg)

 

Terminal Body Weight (g)

Absolute Adrenal (g)

Relative Adrenal (%)

Absolute Kidney (g)

Relative Kidney (%)

Absolute Liver (g)

Relative Liver (%)

1

0

Mean

SD

1979.2

7.3

0.067

0.007

0.034

0.004

1.659

0.104

0.841

0.028

6.465

0.317

3.280

0.140

2

15

Mean

SD

221.4

29.8

0.069

0.007

0.032

0.006

1.819

0.227

0.823

0.043

7.220

1.245

3.250

0.132

3

150

Mean

SD

220.2

11.6

0.074

0.007

0.034

0.004

1.966*

0.142

0.892

0.020

7.997*

0.574

3.632*

0.158

4

750

Mean

SD

218.2

15.2

0.070

0.009

0.032

0.003

2.703*

0.113

0.952*

0.046

9.814*

1.255

4.490*

0.361

Relative organ weight = (absolute organ weight x 100) / terminal body weight (%)

*Indicates a statistically significant difference from the control group (p<0.05)

Applicant's summary and conclusion

Conclusions:
The NOAEL of the test substance was determined to be 15 mg a.i./kg based on liver and kidney weight increases at higher dose levels.
Executive summary:

The oral repeat dose toxicity of the test substance was determined in accordance with the standardised guidelines OECD 407 and EU Method B.7. Groups of six male and six female rats were dosed orally by gavage with 0 (control), 15, 150 or 750 mg a.i./kg of the test substance for 28 consecutive days. Clinical observations, body weights and food consumption were measured throughout the study. At the end of the treatment period, all rats were euthanised and necropsied. Selected organ weights were recorded and tissues were collected for histopathologic evaluation.

There were no mortalities or clinical signs indicative of systemic toxicity in any of the treatment groups. There was a slight decrease in mean body weight and cumulative body weight gain which progressed through the treatment period among males at 750 mg/kg. No treatment-related effects were noted in mean body weight or cumulative body weight gain in females, or food consumption in both sexes.

No treatment-related heamatological effects were noted. Treatment-related changes in clinical chemistry parameters were only observed in males at 750 mg/kg and included: increased alkaline phosphatase (47%), decreased total protein (11%) and globulin (41%) with a corresponding increase in albumin: globulin ratio (83%).

Treatment-related increases in absolute (18 -52%) and relative (11 -47%) liver weights were noted only at 150 and 750 mg/kg in both sexes. Since the liver weight increases at 150 mg/kg were minimal and had no histopathological correlate, the toxicological significance of this finding is unclear and these changes may not represent an adverse effect.

Treatment-related pathologic findings were noted in both sexes only at 750 mg/kg and included: thickened mucosa or raised focus/area in the forestomach and thickening of the glandular mucosa.

Treatment-related microscopic changes were evident in males and/or females only at 750 mg/kg and included: hyperplasia, hyperkeratosis, oedema, inflammation and ulceration/erosion of the forestomach, submucosal oedema of the glandular stomach and diffuse hepatocellular hypertrophy in the liver.

In consideration of the findings, the No-Observed Adverse Effect Level was determined to be 15 mg/kg since the only effect seen at this level was a minimal increase in liver weights without a histopathological correlate. Toxicologically significant liver and kidney weight increases were seen in animals dosed at higher concentrations.