3,4-dihydro-2H-1,4-benzoxazin-6-ol

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:

From 10 november 1988 to 18 april 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

EC Number:

247-415-5

EC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Cas Number:

26021-57-8

Molecular formula:

C8H9NO2

IUPAC Name:

3,4-dihydro-2H-1,4-benzoxazin-6-ol

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

OFA

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Credo, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: males 171 to 203 g and females 130 to 167 g

- Fasting period before study: not specified
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): pellets ad libitum (brand U.A.R. A04 CR)
- Water (e.g. ad libitum): treated tap water ad libitum in auto watering system
- Acclimation period: at least 6 days

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): lighting period from 7h00 to 19h00

IN-LIFE DATES: From: To: not specified

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral, by oesophageal intubation.
Rationale behind oral administration and choice of doses: this route of administration is supposed to assue an absorption at least equal to cutaneous application. The doses were chosen with reference to the expected dosage for human application.

Vehicle:

other: suspended in a hydrogel containing 2% polysorbate 80 in sterile water (containing dimethicone as an anti-foaming agent)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance is suspended in the vehicle daily, just before being sent to the toxicology laboratory for dosing.
The volume of administration will be adjusted, with preference to the weight of animals, twice a week. The animals will be dosed, each day, between 8 and 13 hours, except for major reasons which will be noted in the principal resgister of the study.

DIET PREPARATION : not applicable (gavage)

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified in the study report.
- Concentration in vehicle:
--> Dose group 2: 1 mg/mL
--> Dose group 3: 10 mg/mL
--> Dose group 4: 100 mg/mL
- Amount of vehicle (if gavage): volume of 10 mg/kg bw
- Lot/batch no. (if required): not specified.
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF FORMULATIONS:
-IDENTIFICATION AND CONCENTRATION: once, at the beginning of the stuyd for each dose (sample = 30 mL).
-STABILITY: at the beginning of the study, the stability of the test article was verified only for the highest dose, just after the preparation and after 24 hours (sample = 30 mL).
-HOMOGENEITY: at the beginning of the study, the homogeneity of the suspensions was verified for the highest doses; three samples of 10 mLwere taken, one at te top, middle and bottom of the suspension. All the samples were frozen immediately and stored frozen until analyzed by the test facility.

Duration of treatment / exposure:

30 days for males and 31 days for females.

Frequency of treatment:

Once a day with 24 hours between doses.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose level.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not sepcified in the study report.
- Rationale for animal assignment (random): the animals willl be assigned to groups using a random number generator immediately on arrival. During the period of pretest, animals of groups 1 to 4 will be replaced by animals ofgroup 5 if necessary to produce homogenous body weights or because of poor condition. Groyp 5 animals will also be used to replace those dying through accident during the first two days of the study. The animals not used in these ways will be killed without necropsy on day 3.
- Fasting period before blood sampling for clinical biochemistry: yes
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
- Other:none

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Not specified

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Pretest: up to 5 times per day (days -6, -5, -4, -3, -2 and -1); grace period days: [ -1, +1]
Scheduled Study Days starting on day 1 every day through day 31; grace Period Days:[ -1, +1]

BODY WEIGHT: Yes
- Time schedule for examinations:
Pretest: days -5 and -1; Grace Period Days:[-1, +1]
Scheduled Study Days: days 1, 4, 8 , 11, 15, 18, 22, 25 and 28; Grace Period Days:[-1, +1]

FULL FEEDER WEIGHTS:
Pretest: none
Scheduled Study Days: 1, 4, 8, 11, 15, 18, 22 and 25; Grace Period Days:[ -1, +1]

EMPTY FEEDER/ FEED CONSUMED:
Scheduled Study Days: 4, 8 , 11, 15, 18 , 22, 25 and 28; Grace Period Days:[ -1, +1]

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

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 / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes; examinations will be performed in non anaesthetized animals after instillation in each eye of a drop of Tropicanamide; Time of examinations: 3 to 6 hours after treatment
- Time schedule for examinations:
Scheduled Pretest Days: day -4; Grace Period Days:[-1, +1]
Scheduled Study Days: day 28; Grace Period Days:[-1, +1]
- Dose groups that were examined:
•PRETEST : all the animals
•TEST PERIOD : ALL THE ANIMALS of GROUPS 1 AND 4; in case of abnormality in the animals of group 4, all the animals of grpups 2 and 3 will also be examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29; Grace Period Days:[ -1, +1]. The blood was sampled via the retro-orbital sinus in the Fluothane-Anesthetized animals deprived of food for a period of 16 to 18 hours. Sampling was performed about 24 hours after treatment. 0.5 ml in EDTA for the blood and differential white cell count and 0.5 ml in sodium citrtate 3.8% (dilution 1/10) for the coagulation parameter. Bone marrow was suspended in the foetal calf serum.
- Anaesthetic used for blood collection: Yes (Fluothane)
- Animals fasted: Yes (animals deprived of food for a period of 16 to 18 hours)
- How many animals: 10 animals from each sex from dose groups 1, 2, 3 and 4.
- The following parameters were examined:
BLOOD COUNT: ERYTHROCYTE COUNT, HEMOGLOBIN CONCENTRATION, HEMATOCRIT, MEAN CORPUSCULAR VOLUME, MEAN CORPUSCULAR HGB, MEAN CORPUSCULAR CONCENTRATION, PLATELETS
DIFFERENTIAL WHITE CELL COUNT: LEUKOCYTE, NEUTROPHIL POLYNUCLEARS, EOSINOPHIL POLYNUCLEARS, BASOPHIL POLYNUCLEARS, LYMPHOCYTES, MONOCYTES, TOTAL NEUTROPHIL COUNT, TOTAL LYMPHOCYTES COUNT
COAGULATION: PROTHROMBIN TIME
- The slides for reticulocyte counts and the smears for myelograms were prepared on all animals. The count of reticulocytes and the examination of bone marrow smears were performed only in case of hematological abnormality. This will be the subject of a protocol amendment.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29; Grace Period Days:[ -1, +1]. The blood was sampled via the retro-orbital sinus in the Fluothane-Anesthetized animals deprived of food for a period of 16 to 18 hours. Sampling was performed about 24 hours after treatment. About 2 ml in dry tube.
- Animals fasted: Yes (animals deprived of food for a period of 16 to 18 hours)
- How many animals: 10 animals from each sex from dose groups 1, 2, 3 and 4.
- The following parameters were examined:
Substrate and enzymes: GLUCOSE, UREA, CREATININE, CHOLESTEROL, TRIGLYCERIDES, PROTEIN, ALBUMIN, GLOBULINS, ALBUMIN, ALKALINE PHOSPHATASE, ALANINE AMINOTRANSFERASE, ASPARTATE AMINOTRANSFERASE
Electrolytes: SODIUM, POTASSIUM, CHLORIDE, CALCIUM, PHOSPHORE
- The electrophoresis or serum total bilirubin were carried out in the event of abnormal ALBUMIN/GLOBULIN ratio or Serum hepatic enzymatic level respectively and specifed by protocol amendement.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: day 31; Grace Period Days:[ -1, +1]. After administration of about 10 ml/kg of water by oesophage intubation, the animals were housed individually in diuresis cages with deprivation of food and water. The beginning of collection was about 6 hours after treatment and duration of collection was about 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (deprivation of food and water)
- How many animals: 10 animals from each sex from dose groups 1, 2, 3 and 4.
- The following parameters were examined:
Quantitative analysis: VOLUME, SPECIFIC GRAVITY, PROTEINS (MG/ML), PROTEINS (MG/ANIMAL/16H)
Qualitative analysis (scored): COLOR, GLUCOSE, KETONES, BILE PIGMENTS, UROBILINOGEN, HEMOGLOBIN PIGMENTS, NITRITES
Microscopic analysis (scored): ERYTHROCYTES, LEUKOCYTES, EPITHELIAL CELLS, BLADDER CELLS, KIDNEY CELLS, AMMONIUM MG. PHOS. CRYSTALS, CALCIUM PHOSPHATE CRYSTALS, CALCIUM OXALATE CRYSTALS, HYALINE CASTS, GRANULAR CASTS
- If a discolouration of the urines, due to elimination of the compound is observed, it may be impossible to perform all the scheduled urinary examinations. In that case, these examinations will be cancelled at least in treated groups where the urinary discoulouration precludes them.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

NECROPSY:
- The necropsies were performed in the animals deprived of food and water for a period of 16 to 24 hours. Sacrifice was performed using CO2 inhalation and bleeding.
- All the macroscopically abnormal tissues were collected. The femur was not collected in animals found dead. After the collection, the femurs were sent to the clinical biology laboratory for the preparatoon of bone marrow smears.
- The paired organs were weighted together.
- All the tissues collected were fully processed.

OTHER: None

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organs to weight: LIVER, KIDNEYS, ADRENALS, TESTES (males), OVARIES (females)
Tissues to collect: SPLEEN, AORTA, THYMUS, JEJUNUM, CECUM, SEMINAL VESICLES, VAGINA, ADRENALS, PITUITARY GLAND, BRAIN, SALIVARY GLANDS, LUNGS & BRONCHI, KIDNEYS, URINARY BLADDER, LIVER, HEART, STOMACH, DUODENUM, ILEUM, COLON, TESTES, PROSTATE/VENTRAL, OVARIES, UTERUS, BONE STERNUM, BONE MARROW, STER. BONE MARROW, FEM. BONE MARROW, TRACHEA, ESOPHAGUS, EYES, OPTIC NERVES, MAMMARY GLANDS, SKIN, PANCREAS, MESENTERIC L. N., THYROIDS

HISTOPATHOLOGY: Yes
- All macroscopically abnormal tissues were examined microscopically. For paired organs, both were examined.
- Because of the small degree of development of the mammary gland in male animals, no recut was requested when the tissue was not present on the original slide.
- In the animals of groups 2 and 3, only tissues which show treatment related histological abnormality were examined; this would be the subject of a protocol amendment.
- Tissues for Microscopic examination: SPLEEN, KIDNEYS, URINARY BLADDER, LIVER, HEART, THYMUS, STOMACH, DUODENUM, JEJUNUM, ILEUM, COLON, TESTE, OVARIES, UTERUS, BONE STERNUM, STER.BONE MARROW, ADRENALS, TRACHEA, ESOPHAGUS, PITUITARY GLAND, EYES, OPTIC NERVES, BRAIN, MAMMARY GLANDS, SKIN, SALIVARY GLANDS, PANCREAS, MESENTERIC L.N., LUNGS & BRONCHI, THYROIDS

Optional endpoint(s):

Optional endpoints: No

Other examinations:

None.

Statistics:

If possible, the results are statistically analysed using the Bartlett's TEST followed by the Dunnett's TEST (data homogenous) or by the modiifed test of signficance (data non homogenous):

- The results are given in the form of the mean accompanied by the standard deviation.
- Comparison of the means:
1. Comparison using the pre-test value
"t" test of pairs (t table, Fisher and Yates)
2. Comparison using the control group
-> Bartlett's test (test of the comparison of several variances, CHI-2 table, Fisher and Yates).
-> Dunnett's "t" test (t table for comparison between several treatments and a control).
-> Modified "t" test (t table, Fisher and Yates).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

30 minutes after the first administration, the animals dosed with the 1000 mg/kg/day dose presented hypomotility with ptosis and excess salivation, ataxia was also present in the females animals. The hypomotility was observed for 2 to 5 hours, varying between individual animals. This syndrome, without salivation (except for one animal)was seen again on day 2, when dyspnoea and repetitive movements were also noted in 3 females as well as chromorhinorrhea in most of them. From day 3 onwards, the female animals'coats of this dose group appeared dull and the clinical observations made on the preceding days disappeared. A non dose related chromodacryorrhea was also observed in some animals and alopecia in 3 female animals (2 controls and 1 animal given 10 mg/kg/day) and in 1 male animal (1000 mg/kg/day).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No deaths related to compound administration were reported.
One male animal treated with 10 mg/kg/day died on day 29 during blood sampling.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At the end of the study, a decrease in weight gain of the order of 7.5 %was noted in male animals treated with the 1000 mg/kg/day dose:
- A slowing of bodyweight gain with losses in weight in 4 female animals was observed between days 1 and 4 in the animals treated with the 1000 mg/kg/day dose, then the weight gain continued normally until day 15.
- After day 18, again a slight slowing in weight gain was noted in the males but for all the doses.
- Loss of weight was recorded between days 15 and 18 in 5 female animals in the same cage.

Animal mean body weights (Table 1) and the variations in percentage compared with day 1 (Table 2) are presented in Section "any other information incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of the high dose animals was clearly decreased between days 01 and 04 compared with the control animals (22 % in the males and 28 % in the females):
- A marked decrease in food consumption was noted at the beginning of the study in animals of both sexes receiving the 1000 mg/kg/day. Slight variations were then observed, mainly in the female animals treated with the high dose.

Average feed consumed per day (Table 3) and variations in percentage compared with food consumption of the control animals (Table 4) are presented in Section "any other information incl. tables".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormality attributable to compound administration was observed after 4 weeks of treatment in the animals given the 1000 mg/kg/day dose.

Haematological findings:

no effects observed

Description (incidence and severity):

No significant change was noted in the results of haematological examination. In the male animals treated with the 1000 mg/kg/day dose, a slight increase in the number of neutrophils (x 1.8) was noted.

Summary for blood counts (Table 5), Differential white cells (Table 6) and Coagulation (Table 7) are presented in Section "any other information incl. tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

As far as clinical chemistry was concerned, only the increase in triglycerides (65 %) observed in the female animals of the high dose group appeared to be outside the laboratory norms. A dark coloration of the urine and an increase in urinary proteins was noted in males (x 1.9) and females (x 2.2)of this same group:
- At the 100 and 1000 mg/kg/day doses, a decrease in transaminase ASAT was observed in the males of- 17 % and - 22 % and in the females of 14 % and - 31 %. Moreover, in these two female groups, the alkaline phosphatase level was also decreased by - 24 % and - 28 %.
- At the high dose, in the male animals, transaminase ALAT was slightly raised (x 1.2); triglycerides were decreased in the males by 28 %-and increased in the females by 65 %.
- The phosphorus level was decreased by about 15 % in the males treated with doses of 100 and 1000 mg/kg/day. In the variations mentioned above, the changes in enzyme activity were within physiological limits the level of triglycerides in the female animals was above the upper limit for the species but did not appear to have a pathological nature.
- The other changes noted were considered to be fortuitous and concerned :
--> a slight decrease in serum cholesterol of - 15 % and - 11 % in the males treated at doses of 10 and 100 mg/kg/day,in the females, a minimal rise in proteins and albumin of the order of + 5 % at the 10 mg/kg/day dose and a decrease in globulins of - 6.9 % at the dose of 100 mg/kg/day, the variations in serum potassium level of + 0.5 m Eq/1 in the females of the 100 mg/kg/day dose group and + 0.3 m Eq/1 in the males of the 1000 mg/kg/day dose.

Summary for substrate and enzymes results (Table 8) and Summary for electrolytes results (Table 9) are presented in Section "any other information incl. tables".

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of the results demonstrated a dark coloration and a slight acidification of the urine (males : - 0.7 of a unit ; females : - 0.5 of a unit) as well as an increase in the amount of proteins (x 1.9) in males and x 2.2 in the females) in the 1000 mg/kg/day dose groups.

Urinalysis results – Summary for quantitative analysis (Table 10) are presented in Section "any other information incl. tables".

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Examination of organ weights only showed changes in the high dose (1000 mg/kg/day dose):
- an increase in relative liver weight of 25 % in males, an increase of 17 % absolute liver weight and 22 % relative liver weight in females,
- an increase in relative kidney weight of 13 % and relative testes weight of 11 % in the males. The increase in organ weights noted in this group could be related to the decrease in bodyweight.

Absolute organ weights results (in grams) (Table 11) and Relative organ weights results (Table 12) are presented in Section "any other information incl. tables".

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination did not reveal any abnormality attributable to compound administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination demonstrated changes in the kidney in the males which were attributed to treatment. They were characterised by changes in the cortical tubules, the frequency and severity of which appeared to be related to the administered dose (epithelial necrosis, deposits of anhistic acidophilic substance, basophilia, dilatation). The low dose (10 mg/kg/day) corresponded to the dose which did not induce an adverse effect, the changes observed in this group being approximately comparable to those in the controls. In the females, no compound-related abnormality was observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other: no signs of toxicity at 10 mg/kg/day.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1. Animal mean body weights in grams

Groups	Day of study
		-5	-1	1	4	8	11	15	18	22	25	28
	Male animals
1	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	125.7	170,6	178.9	200.8	232.2	258.8	285.4	309.9	334.0	347.4	361.6
	SDEVS	16.7	4.4	4.4	5.7	7.4	8.3	14.0	13.0	18.6	20.8	22.3
2	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	145.8*	182.6*	188.0*	206.5	238.5	264.3	291.2	308.7	329.7	339.3	355.4
	SDEVS	5.4	4.9	4.9	6.7	7.8	10.9	11.2	14.8	16.1	17.2	17.7
3	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	146.5*	180.7*	187.2*	207.3	237.8	267.1	296.7	312.1	334.9	345.4	358.3
	SDEVS	6.5	7.5	9.3	10.3	13.9	14.9	15.9	20.1	21.5	23.0	26.7
4	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	129.9	174.2	181.4	188.8*	220.7*	248.0	274.7	290.7*	311.4*	321.6*	334.6*
	SDEVS	21.8	7.4	6.0	5.2	4.8	6.6	10.2	12.2	19.3	22.9	26.1
	Female animals
1	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	106.8	143.5	147.9	157.8.	171.4	184.6	202.4	214.2	224.5	228.2	237.7
	SDEVS	19.1	9.4	11.1	11.7	14.2	14.1	16.0	17.4	19.4	18.8	23.4
2	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	106.4	146.1	148.2	161.1	174.8	189.3	209.3	218.1	227.9	233.4	240.4
	SDEVS	19.0	7.9	6.9	8.5	9.5'	11.6'	13.0	12.6	14.4	15.9	19.4
3	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	93.1	139.5	143.2	159.0	174.1	185.9	202.9	214.5	224.3	230.6	238.3
	SDEVS	12.0	6.8	7.8	7.7	9.0	12.5	13.3	15.5	16.4	16.7	18.5
4	(N)	10	10	10	10	10	10	10	10	10	10	10
	Means	112.4	145.1	150.1	152.0	173.3	185.9	203.3	202.5	221.7	224.4	232.5
	SDEVS	19.1	13.6	13.2	16.8	14.5'	13.3	14. 1	20.5	13.1	12.5	12.2

(N): number of animals

* = Mean value ofgroup was significantly different from control atp=0.05 with Dunnett’s Test of significance

 

 

Table 2. Bodyweight variations in percentage compared with day 1

Days	4	8	15	22	28
Males
Controls	+ 12	+ 30	+ 60	+ 87	+ 102
10 mg/kg/day	+ 10	+ 27	+ 55	+ 75	+ 89
100 mg/kg/day	+ 11	+ 27	+ 58	+ 79	+ 91
1000 mg/kg/day	+ 4	+ 22	+ 51	+ 72	+ 84
Females
Controls	+ 7	+ 16	+ 37	+ 52	+ 61
10 mg/kg/day	+ 9	+ 18	+ 41	+ 54	+ 62
100 mg/kg/day	+ 11	+ 22	+ 42	+ 57	+ 66
1000 mg/kg/day	+ 1	+ 15	+ 35	+ 48	+ 55

 

Table 3. Average feed consumed/day of study in grams

Group(s)	Day of study
		4	8	11	15	18	22	25	28
	Male animals
1	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		23.20	24.50	26.80	23.65	33.13	27.85	29.67	27.20
		24.93	25.30	27.13	27.40	28.60	25.85	27.13	24.87
	Means	24.07	24.90	26.97	25.52	30.87	26.85	28.40	26.03
2	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		23.40	24.80	27.53	25.40	29.40	26.15	27.13	25.73
		23.13	25.40	26.93	26.50	29.33	25.40	26.47	25.40
	Means	23.27	25.10	27.23	25.95	29.37	25.77	26.80	25.57
3	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		22.73	24.55	26.80	26.65	29.47	26.25	29.20	25.93
		22.93	23.50	26.93	27.80	27.80	25.15	27.07	26.53
	Means	22.83	24.03	26.87	27.23	28.63	25.70	28.13	26.23
4	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		18.60	23.50	25.80	26.75	28.80	26.60	29.00	25.20
		18.80	23.00	26.80	26.90	28.93	26.90	28.47	27.40
	Means	18.70	23.25	26.30	26.82	28.87	26.75	28.73	26.30
	Female animals
1	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		17.67	18.85	20.33	19.65	21.47	20.25	21.00	20.87
		19.07	18.20	20.13	21.15	23.07	20.30	21.80	21.33
	Means	18.37	18.52	20.23	20.40	22.27	20.28	21.40	21.10
2	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		17.60	17.10	18.73	18.75	20.53	18.60	20.20	18.40
		19.13	19.05	21.53	21.75	23.40	21.70	23.27	21.87
	Means	18.37	18.08	20.13	20.25	21.97	20.15	21.73	20.13
3	No. Cages (No. animals)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)	2(10)
		20.67	19.05	19.80	19.90	23.67	20.55	20.47	19.13
		18.67	19.15	19.80	19.50	22.53	19.65	19.60	19.87
	Means	19.67	19.10	19.80	19.70	23.10	20.10	20.03	19.50
4	No. Cages (No. animals)	2(10)	2(10)	2(10)	1(5)	2(10)	2(10)	2(10)	2(10)
		13.47	17.80	19.20	18.80	17.27	20.00	21.40	19.53
		12.93	17.65	18.93		21.67	19.20	20.33	18.87
	Means	13.20	17.72	19.07	18.80	19.47	19.60	20.87	19.20

 

Table 4. Food consumption variations in percentage compared with food consumption of the control animals

Days	1 to 4	4 to 8	8 to 15	15 to 22	22 to 28
Males
10 mg/kg/day	- 3	+ 1	+ 1	- 4	- 4
100 mg/kg/day	- 5	- 3	+ 3	- 6	0
1000 mg/kg/day	- 22	- 7	+ 1	- 4	+ 1
Females
10 mg/kg/day	0	- 2	- 1	- 1	- 2
100 mg/kg/day	+ 7	+ 3	- 3	+ 2	- 7
1000 mg/kg/day	- 28	- 4	- 7	- 8	- 6

 

Table 5. Haematological examination results – Summary for blood counts

	Mean Blood count values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units						Males
ERYTHROCYTE COUNT E6/MM3	29	8.05	0.32	9	8.02	0.34	10	7.84	0.29	10	7.96	0.30	10
HEMOGLOBIN CONCENTRATION G/DL	29	15.77	0.69	9	15.80	0.72	10	15.70	0.56	10	16.04	0.89	10
HEMATOCRIT %	29	48.12	2.49	9	48.06	2.10	10	46.94	1.71	10	48.43	1.86	10
MEAN CORPUSCULAR VOLUME U3	29	59.78	1.39	9	60.00	2.16	10	59.80	1.14	10	60.80	1.69	10
MEAN CORPUSCULAR HGB UUG	29 (n)	19.60	0.50	9	19.74	1.12	10	20.03	0.51	10	20.16	0.90	10
MEAN CORPUSCULAR HGB CONCENT G/DL	29	32.79	0.93	9	32.89	1.25	10	33.46	1.05	10	33.11	0.97	10
PLATELETS E3/MM3	29	887.56	81.56	9	843.60	84.11	10	897.40	120.09	10	840.60	84.96	10
	Females
ERYTHROCYTE COUNT E6/MM3	29	7.48	0.39	9	7.93*	0.30	10	7.57	0.31	10	7.16	0.41	10
HEMOGLOBIN CONCENTRATION G/DL	29	14.96	0.81	9	15.74	0.71	10	14.55	0.80	10	14.32	0.83	10
HEMATOCRIT %	29	47.77	2.38	9	50.25	2.36	10	48.05	2.01	10	46.60	2.74	10
MEAN CORPUSCULAR VOLUME U3	29	64.00	1.66	9	63.40	1.71	10	63.40	1.78	10	65.10	1.20	10
MEAN CORPUSCULAR HGB UUG	29 (n)	20.01	0.95	9	19.84	0.77	10	19.22	0.59	10	19.99	0.35	10
MEAN CORPUSCULAR HGB CONCENT G/DL	29	31.31	0.79	9	31.32	0.83	10	30.27*	0.60	10	30.72	0.70	10
PLATELETS E3/MM3	29	844.67	83.57	9	849.50	150.79	10	829.30	153.76	10	902.00	109.95	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 6. Haematological examination results – Summary for Differential white cells

	Mean Diff. White cell count values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units							Males
LEUKOCYTE COUNT E3/MM3	29	8.0	1.66	9	7.51	1.15	10	8.26	1.54	10	7.80	1.39	10
TOTAL NEUTROPHIL COUNT /MM3	29	602.44	325.50	9	744.00	248.17	10	663.20	266.04	10	1059.00**	341.22	10
TOTAL LYMPHOCYTE COUNT /MM3	29	7085.39	1476.19	9	6555.00	1192.52	10	7412.80	1508.39	10	6645.70	1184.37	10
NEUTROPHIL POLYNUCLEARS %	29	7.56	3.9B	9	10.15	4.06	10	8.05	3.06	10	13.40**	3.63	10
EOSINOPHIL POLYNUCLEARS %	29	1.56	1.04	9	1.10	0.99	10	0.95	0.96	10	0.50*	0.71	10
BASOPHIL POLYNUCLEARS %	29	0.00	0.00	9	0.00	0.00	10	0.00	0.00	10	0.00	0.00	10
LYMPHOCYTES %	29	88.17	4.05	9	86.90	4.58	10	89.55	4.04	10	85.40	4.03	10
MONOCYTES %	29	2.72	1.82	9	1.80	1.69	10	1.45	1.50	10	0.70**	0.95	10
	Females
LEUKOCYTE COUNT E3/MM3	29	5.32	1.05	9	6.03	1.48	10	5.15	1.17	10	6.01	1.63	10
TOTAL NEUTROPHIL COUNT /MM3	29	641.22	333.13	9	438.90	193.46	10	436.60	163.65	10	552.10	161.42	10
TOTAL LYMPHOCYTE COUNT /MM3	29	4600.44	732.62	9	5528.00	1494.88	10	4624.60	973.10	10	5372.90	1544.11	10
NEUTROPHIL POLYNUCLEARS %	29	11.56	3.91	9	7.60*	3.81	10	8.30	1.77	10	9.50	2.95	10
EOSINOPHIL POLYNUCLEARS %	29(n)	1.33	0.50	9	1.20	1.81	10	1.40	1.26	10	1.20	1.23	10
BASOPHIL POLYNUCLEARS %	29	0.00	0.00	9	0.00	0.00	10	0.00	0.00	10	0.00	0.00	10
LYMPHOCYTES %	29	87.00	4.21	9	91.20*	3.91	10	90.10	2.60	10	89.20	2.78	10
MONOCYTES %	29	0.11	0.33	9	0.00	0.00	10	0.20	0.42	10	0.10	0.32	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 7. Haematological examination results – Summary for Coagulation

	Mean Coagulation values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units							Males
PROTHROMBIN Time S	29	15.79	0.54	8	15.79	0.67	10	16.02	0.45	10	15.62	0.46	10
	Females
PROTHROMBIN Time S	29	16.34	0.47	9	16.43	0.42	10	16.51	0.59	10	16.54	0.45	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 8. Biochemical examination – Summary for substrate and enzymes results

	Mean substrate and enzymes values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units							Males
GLUCOSE g/L	29	1.12	0.20	10	1.16	0.12	10	1.2	0.14	10	1.01	0.10	10
UREA g/L	29	0.35	0.05	10	0.36	0.06	10	0.33	0.05	10	0.37	0.04	10
CREATININE mg/L	29	4.81	1.07	10	4.80	1.06	10	4.65	1.08	10	4.61	0.94	10
CHOLESTEROL g/L	29(n)	0.94	0.10	10	0.80**	0.09	10	0.84*	0.09	10	0.93	0.24	10
TRIGLYCERIDES g/L	29	0.93	0.27	10	o. 76	0.16	10	0.82	0.20	10	0.67*	0.21	10
PROTEIN g/L	29	62.20	1.93	10	62.30	1.49	10	63.40	2.67	10	63.50	3.41	10
ALBUMIN g/L	29	34.00	1.05	10	34.10	0.99	10	34.40	1.07	10	35.00	1.63	10
GLOBULINS g/L	29	28.20	1.81	10	28.20	1.32	10	29.00	2.00	10	28.50	2.07	10
ALBUMIN/GLOBULINS	29	1.21	0.09	10	1.21	0.07	10	1.19	0.07	10	1.23	0.07	10
ALKALINE PHOSPHATASE UI/L	29	321.80	102.67	10	343.90	64.05	10	306.70	72.69	10	276.20	46.32	10
ALANINE AMINOTRANSFERASE UI/L	29	22.00	4.40	10	22.80	2.25	10	21.60	3.37	10	27.20*	4.66	10
ASPARTATE AMINOTRANSFERASE UI/L	29(n)	66.40	12.52	10	60.80	6.73	10	55.40*	5.72	10	52.00**	4.52	10
	Females
GLUCOSE g/L	29	1.07	0.05	10	1.11	0.08	10	1.12	0.07	10	1.08	0.10	10
UREA g/L	29	0.46	0.08	10	0.53	0.06	10	0.50	0.05	10	0.48	0.08	10
CREATININE mg/L	29	6.60	1.12	10	6.72	0.98	10	6.80	0.97	10	6.23	0.81	10
CHOLESTEROL g/L	29(n)	1.02	0.28	10	1.05	0.10	10	0.92	0.14	10	1.22	0.29	10
TRIGLYCERIDES g/L	29(n)	0.62	0.14	10	0.50	0.10	10	0.55	0.11	10	1.02*	0.44	10
PROTEIN g/L	29	64.40	2.17	10	67.60*	2.12	10	62.10	2.73	10	64.00	4.14	10
ALBUMIN g/L	29	35.50	1.72	10	37.60*	1.08	10	35.20	1.55	10	36.40	2.46	10
GLOBULINS g/L	29	28.90	0.99	10	30.00	1.63	10	26.90*	1.85	10	27.60	2.27	10
ALBUMIN/GLOBULINS	29	1.23	0.07	10	1.26	0.07	10	1.32	0.09	10	1.32*	0.09	10
ALKALINE PHOSPHATASE UI/L	29	256.90	49.27	10	242.50	52.22	10	195.00*	29.25	10	184.70*	75.64	10
ALANINE AMINOTRANSFERASE UI/L	29	20.10	2.33	10	20.00	2.87	10	18.60	3.10	10	22.00	7.07	10
ASPARTATE AMINOTRANSFERASE UI/L	29	69.00	5.54	10	63.70	7.12	10	59.40**	6.85	10	47.40**	4.20	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 9. Biochemical examination – Summary for electrolytes results

	Mean electrolytes values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units							Males
SODIUM MEQ/L	29	145.70	0.82	10	146.00	1.05	10	145.80	1.03	10	145.50	1.08	10
POTASSIUM MEQ/L	29	5.08	0.29	10	5.05	0.22	10	5.09	0.25	10	5.39*	0.23	10
CHLORIDE MEQ/L	29(n)	104.70	1.06	10	104.90	2.28	10	106.40**	0.84	10	105.90	1.45	10
CALCIUM MEQ/L	29	101.80	1.99	10	100.40	3.47	10	102.60	3.41	10	103.80	3.22	10
PHOSPHORE MEQ/L	29	100.80	6.56	10	98.80	5.71	10	86.60••	6.90	10	86.10••	4.86	10
	Females
SODIUM MEQ/L	29	144.50	1.51	10	144.50	1.08	10	144.80	1.40	10	144.50	1.65	10
POTASSIUM MEQ/L	29	4.82	0.18	10	4.97	0.22	10	5.34**	0.38	10	5.02	0.33	10
CHLORIDE MEQ/L	29	106.00	1.15	10	106.00	1.15	10	106.70	1.42	10	106.60	0.84	10
CALCIUM MEQ/L	29	101.30	2.95	10	103.20	1.62	10	101.90	2.60	10	105.20**	2.30	10
PHOSPHORE MEQ/L	29	83.10	7.32	10	82.60	4.84	10	78.20	10.57	10	77.50	4.90	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 10. Urinalysis results – Summary for quantitative analysis

	Mean quantitative analysis values
		Control			Group 2			Group 3			Group 4
	Study day	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Parameter /Units							Males
VOLUME (ml/animal/16h) mL/16h	31	9.45	2.05	10	7.58	3.68	9	8.94	2.49	10	11.61	2.65	10
SPECIFIC GRAVITY	31(n)	1.02	0.00	10	1.04*	0.01	9	1.03*	0.00	10	1.03**	0.00	10
pH	31	7.58	0.46	10	7.94	0.52	9	7.80	0.49	10	6.85**	0.43	10
PROTEINS (mg/mL)	31	0.34	0.10	10	0.39	0.14	9	0.36	0.11	10	0.54**	0.16	10
PROTEINS (mg/animal/16h) mg/16h	31	3.23	1.13	10	2.56	0.82	9	3.05	0.93	10	6.06**	1.39	10
	Females
VOLUME (ml/animal/16h) mL/16h	31	6.76	2.52	10	6.96	3.54	10	7.83	2.75	10	8.28	2.46	10
SPECIFIC GRAVITY	31	1.03	0.00	10	1.03	0.01	10	1.03	0.01	10	1.03	0.01	10
pH	31	6.99	0.32	10	6.83	0.22	10	6.81	0.45	10	6.49*	0.43	10
PROTEINS (mg/mL)	31	0.10	0.05	10	0.10	0.04	10	0.14	0.04	10	0.16**	0.03	10
PROTEINS (mg/animal/16h) mg/16h	31	0.59	0.24	10	0.81	0.50	10	1.06	0.50	10	1.28**	0.46	10

Controls from group(s):1

Data homogenous by Bartlett’s Test; Test for significance is Dunnett’s Test

(n) Data non-homogeneous byBarlett’sTests; Modified t Test of significance

* Indicates group mean is significantly different from control at level p=0.05

** Indicates group mean is significantly different from control at level p=0.01

 

Table 11. Absolute organ weights results (in grams) – Group comparison statistics for absolute organ weights

	Controls from group 1	Data homogeneous by HOMOGENEOUS BY BARTLETT'S TEST (DUNNETT'S t TEST)
Organ: Liver
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	10.484	9.571	10.598	11.566
Standard deviation	1.130	0.733	0.823	1.231
Group Diff. P=0.05		1.136	1.106	1.106
Group Diff. P=0.01		1.442	1.404	1.404
ANALYSIS OF VARIANCE: F RATIO = 6.23 DF = 3/ 35 F PROBABILITY = 0.002
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	6.823	7.066	7.090	7.997
Standard deviation	0.730	0.730	0.884	0.889
Group Diff. P=0.05		0.891	0.891	0.891*
Group Diff. P=0.01		1.130	1.130	1.130*
ANALYSIS OF VARIANCE: F RATIO = 4.04 DF = 3/ 36 F PROBABILITY = 0.014
Organ: Kidneys
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	2.548	2.546	2.542	2.551
Standard deviation	0.224	0.145	0.156	0.173
Group Diff. P=0.05		0.201	0.195	0.195
Group Diff. P=0.01		0.255	0.248	0.248
ANALYSIS OF VARIANCE: F RATIO = 0.01 DF = 3/ 35 F PROBABILITY = 0.997
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	1.774	1.738	1.717	1.763
Standard deviation	0.266	0.154	0.184	0.122
Group Diff. P=0.05		0.208	0.208	0.208
Group Diff. P=0.01		0.263	0.263	0.263
ANALYSIS OF VARIANCE: F RATIO = 0.18 DF = 3/36 F PROBABILITY = 0.907
Organ: Adrenals
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	0.059	0.054	0.056	0.057
Standard deviation	0.009	0.007	0.007	0.005
Group Diff. P=0.05		0.008	0.008	0.008
Group Diff. P=0.01		0.010	0.010	0.010
ANALYSIS OF VARIANCE: F RATIO =0.83 DF = 3/35 F PROBABILITY = 0.488
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	0.074	0.070	0.068	0.072
Standard deviation	0.014	0.010	0.006	0.006
Group Diff. P=0.05		0.012	0.011	0.011
Group Diff. P=0.01		0.018	0.016	0.016
ANALYSIS OF VARIANCE: F RATIO =0.78 DF = 3/36 F PROBABILITY = 0.518
Organ: Testis
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	3.715	3.554	3.700	3.634
Standard deviation	0.220	0.152	0.199	0.264
Group Diff. P=0.05		0.242	0.235	0.235
Group Diff. P=0.01		0.307	0.298	0.298
ANALYSIS OF VARIANCE: F RATIO =1.10 DF = 3/35 F PROBABILITY = 0.362
Organ: Ovaries
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	0.140	0.135	0.141	0.136
Standard deviation	0.019	0.022	0.016	0.017
Group Diff. P=0.05		0.020	0.020	0.020
Group Diff. P=0.01		0.026	0.026	0.026
ANALYSIS OF VARIANCE: F RATIO =0.23 DF = 3/36 F PROBABILITY = 0.875

Note:a*indicates group mean is significantly different from control at level of significance shown.

 

Table 12. Relative organ weights results – Group comparison statistics for % organ to body weight ratio

	Controls from group 1	Data homogeneous by HOMOGENEOUS BY BARTLETT'S TEST (DUNNETT'S t TEST)
Organ: Liver
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	3.129	2.94B	3.202	3.904
Standard deviation	0.206	0.145	0.118	0.368
Group Diff. P=0.05		0.186	0.170	0.303*
Group Diff. P=0.01		0.268	0.245	0.435*
ANALYSIS OF VARIANCE: F RATIO = 31.76 DF = 3/ 35 F PROBABILITY = 0.000
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	3.095	3.186	3.254	3.772
Standard deviation	0.148	0.250	0.212	0.322
Group Diff. P=0.05		0.265	0.265	0.265*
Group Diff. P=0.01		0.336	0.336	0.336*
ANALYSIS OF VARIANCE: F RATIO = 15.85 DF = 3/ 36 F PROBABILITY = 0.000
Organ: Kidneys
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	10	0.784	0.769	0.863
Standard deviation	0.762	0.024	0.026	0.070
Group Diff. P=0.05	0.042	0.035	0.035	0.058*
Group Diff. P=0.01	10	0.051	0.051	0.084*
ANALYSIS OF VARIANCE: F RATIO = 10.92 DF = 3/ 35 F PROBABILITY = 0.000
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	0.802	0.784	0.790	0.833
Standard deviation	0.059	0.062	0.054	0.043
Group Diff. P=0.05		0.060	0.060	0.060
Group Diff. P=0.01		0.077	0.077	0.077
ANALYSIS OF VARIANCE: F RATIO = 1.53 DF = 3/36 F PROBABILITY = 0.222
Organ: Adrenals
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	0.017	0.016	0.017	0.019
Standard deviation	0.002	0.002	0.002	0.002
Group Diff. P=0.05		0.002	0.002	0.002
Group Diff. P=0.01		0.003	0.003	0.003
ANALYSIS OF VARIANCE: F RATIO =3.04 DF = 3/35 F PROBABILITY = 0.041
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	0.034	0.031	0.031	0.034
Standard deviation	0.004	0.004	0.003	0.005
Group Diff. P=0.05		0.005	0.005	0.005
Group Diff. P=0.01		0.006	0.006	0.006
ANALYSIS OF VARIANCE: F RATIO =1.22 DF = 3/36 F PROBABILITY = 0.316
Organ: Testis
Males
Group	Control	2	3	4
Number/Group	10	9	10	10
Mean	1.114	1.097	1.123	1.233
Standard deviation	0.090	0.059	0.101	0.157
Group Diff. P=0.05		0.122	0.119	0.119*
Group Diff. P=0.01		0.155	0.151	0.151
ANALYSIS OF VARIANCE: F RATIO =3.21 DF = 3/35 F PROBABILITY = 0.034
Organ: Ovaries
Females
Group	Control	2	3	4
Number/Group	10	10	10	10
Mean	0.064	0.061	0.065	0.065
Standard deviation	0.006	0.008	0.005	0.009
Group Diff. P=0.05		0.008	0.008	0.008
Group Diff. P=0.01		0.010	0.010	0.010
ANALYSIS OF VARIANCE: F RATIO =0.6 DF = 3/36 F PROBABILITY = 0.620

Note:a*indicates group mean is significantly different from control at level of significance shown.

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

In this study, daily oral administration of hydroxybenzomorpholine to rats for 4 weeks did not produce any signs of toxicity at 10 mg/kg/day. The main changes were microscopic findings in the kidneys observed with a dose-related incidence and severity at 100 and 1000 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg/day.

Executive summary:

The test substance, suspended in a hydrogel containing 2% polysorbate 80 in sterile water (containing dimethicone as an anti-foaming agent), was administered by gavage once daily to groups of Sprague Dawley rats (10/sex) for 30 days (males) and 31 days (females). The dose levels were 10, 100 and 1000 mg/kg bw. The control group (10/sex) received the vehicle alone.

All animals were killed at the end of the study.

All animals were observed twice daily for mortality and clinical signs. Body weights and food consumption were recorded individually at weekly intervals. Ophthalmoscopic examination was performed on day 0 and at termination in the control and high dose group animals only. Blood samples were taken from all animals at day 0 and at termination for haematological and clinical chemistry investigations. Urine samples were collected on day 0 and during week 4. Organ weights were recorded. Macroscopic and histopathologic examination of tissues was undertaken.

Results

No treatment related mortalities were reported. Abnormalities reported in animals treated with 1000 mg/kg bw included; lethargy and excessive salivation, decreased body weight in males, decreased food consumption in the first three days of treatment in both sexes, a slight increase in the number of neutrophils in males, a 65 % increase in triglycerides in females, dark discolouration and slight acidification of the urine and an increase in urinary proteins. The high dose group also showed an increase in both relative and absolute liver, kidney and testis weights.

There were no overt or biochemical signs of toxicity in animals treated with either 10 or 100 mg/kg bw.

Histological examination of the kidneys showed dose-related epithelial necrosis, basophilia and tubular dilation in the cortical tubules in male rats treated with 100 and 1000 mg/kg bw. No effects were seen in animals treated with 10 mg/kg bw per day. No histopathological effects were seen in female rats.

Conclusion

In this study, daily oral administration of hydroxybenzomorpholine to rats for 4 weeks did not produce any signs of toxicity at 10 mg/kg/day. The main changes were microscopic findings in the kidneys observed with a dose-related incidence and severity at 100 and 1000 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg/day.