Mecoprop

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 1977 to 20 December 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

no

Remarks:

The performance of this study pre-dates the inception of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Mecoprop

EC Number:

230-386-8

EC Name:

Mecoprop

Cas Number:

7085-19-0

Molecular formula:

C10H11ClO3

IUPAC Name:

2-(4-chloro-2-methylphenoxy)propanoic acid

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: about 4-5 months old
- Weight at study initiation: females: 6.3-6.4 kg, males: 7.6-8.1 kg
- Housing: The dogs were divided into 3 test groups of 4 males and 4 females and one control group of 4 males and 4 females each. They were individually housed in indoor cages. The dog and
cage number were indicated on each cage.
- Diet: All dogs received daily a weighed portion of food in an amount of 35 g/kg body weight during the first two weeks, and 40 g/kg body weight during the remainder of the test period. The dogs were fed a basal diet of the following percentage composition: instant wheat product 25, beet pulp 5,
instant oat product 25, vitamin B preparation 0.2, fish meal 10, brewer's yeast 3, meat scraps 10, vitamin AD3EK preparation 0.3, skimmed milk powder 5, NaCl + trace minerals 0.5, soya bean oil meal 8, soya bean oil 5, grass meal 3. Analyses are made for nutrients and contaminants.
- Water: unfluorised tap water was always available
- Acclimation period: Two weeks. Animals received an oral anthelmintic treatment (piperazine adipate, 200 mg/kg) twice during the quarantine period.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: molasses/water

Details on oral exposure:

DIET PREPARATION
- The test material was administered by admixture of the test material in the diet. To prepare the test diets firstly a premix was made by mixing 100 g test material in 2000 g basal diet in a Stephan cutter for 2 minutes. The test diets were prepared by diluting appropriate quantities of the premix with basal diet and a molasses/water mixture (1:1). Homogeneity of the diets was achieved by mixing for two minutes in a Lodige mixer. The test diets were mixed in the sequence of increasing concentrations of the test material in the diet.
- Rate of preparation of diet: The diets were freshly prepared at the initiation of the study and then at approximately biweekly intervals.
- Storage temperature of food: The diets were stored at room temperature and were identified by their group number.
- The control group received the basal diet without the test material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The actual levels of the test material in the diets were determined by analyses of the batches of each of the diets prepared in weeks 1, 6 and 12.
- The analyses were made after a storage period of at most 2 weeks.
- The concentrations of the test material recovered in the diet at the different stages were well in line with the nominal concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily (continuous in diet)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

16 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

64 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Four animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose-levels were selected on the basis of the results of a previous range-finding study, in which levels of up to 32 mg/kg body weight/day did not induce any noticeable untoward effect.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS FOR SIGNS OF TOXICITY: Yes
- Health, condition and behaviour of all dogs were checked daily. Special attention was paid to the buccal mucosa and eyelids. All signs of reaction to treatment were recorded.

BODY WEIGHT: Yes
- The weight of each dog was recorded twice weekly for one week pre-dosing and for the first four weeks on test, and then weekly. The weighing’s were generally done when the animals had food available.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each dog was recorded daily and the mean weekly intake per dog was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- At the beginning and at weeks 7 and 13 the eyes of all dogs were examined by indirect ophthalmoscopy.

HAEMATOLOGY: Yes
- Each dog was examined at the beginning and at week 6 and 12. Examinations included:
1.Haemoglobin concentrations - by cyanmethemoglobin method of Van Kampen and Zijlstra (Clin. Chim. Acta 6 (1961) 538-544).
2.Packed cell volume - as microhaematocrit.
3.Erythrocyte count - by Coulter Counter.
4.Leucocyte count - by Coulter Counter.
5. Differential leucocyte count - by direct visual count of smear after May-Griinwald-Giemsa staining.
6.Platelet count - by Coulter Counter.
7. Blood clotting time - using Normotest reagents (Nyegard and Co., Oslo, Norway).

CLINICAL CHEMISTRY: Yes
- Each dog was examined at the beginning and at week 7 and 13. Examinations included:
1. GPT - by Reitman and Frankel (Am. J. Clin. Path. 28 (1957) 56-63).
2. GOT - by Reitman and Frankel.
3. AP - by Bessey, Lowry and Brock (J. Biol. Chem. 164 (1946) 321-329).
4. Total protein - by biuret reaction.
5. Albumin - by Doumas, et al. (Clin. Chim. Acta 31 (1971) 87-96).
6. Serum electrophoresis - by agar gel electrophoretic method on microscopic slides of Wieme.
7. Urea nitrogen - by the automated phenazone/diacetyl monoxime technique of Ceriotti and Spandrio (Clin. Chim. Acta 11 (1965) 519-522).
8. Fasting plasma glucose - by Technicon Auto-Analyzer, method N-9a.
9. Total bilirubin - according to the method of Nederlands Normalisatie Instituut.
10. Serum electrolytes Na+ and K+ - by Paschen and Fuchs (Clin. Chim. Acta 35 (1971) 401-408).
11. OCT - by Ohshita (Clin. Chim. Acta 67 (1976) 145-152).
12. Creatinine - by Jaffe-reaction (In: Gorter en De Graaff, Klinische Diagnostiek).

URINALYSIS: Yes
- Each dog was examined at the beginning and at week 6 and 12. Individual urine samples were collected by catheterisation after overnight deprivation of water and food and examined for:
1. Specific gravity - by refractometer.
2. Electrolytes Na+ and K+ - method of Paschen and Fuchs (Clin. Chim. Acta 35 (1971) 401-408). The following semi-quantitative tests were made:
1. pH - Labstix
2. Protein - Labstix
3. Sugar - Labstix
4. Occult blood - Labstix
5. Ketones - Labstix
6. Bilirubin - Urobilistix
7. Microscopy of the sediment - after centrifugation at 3,000 rpm for 3 minutes. Deposits were examined for: erythrocytes, leucocytes, epithelial cells, crystals, casts, bacteria, sperm cells, and worm eggs.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On completion of 91 days of treatment, all surviving dogs were anaesthetised by intravenous administration of Nembutal and exsanguinated by cutting the carotid artery. The dogs were subjected to a thorough autopsy.
- The following organs from all animals were dissected free of fat and weighed: liver, heart, adrenals, kidneys, brain, pituitary, spleen, lungs, testicles, thymus, thyroid, ovaries and prostate.
- Samples of the following tissues from all dogs were preserved in a 4 per cent neutral, phosphate-buffered formaldehyde solution: liver, kidneys, spleen, jejunum, ileum, caecum, thymus, heart, brain, lung, thyroid, adrenals, pituitary, testicles, ovaries, prostate, salivary glands (3), trachea, diaphragm, gall bladder, buccal mucosa, lymph nodes(cervical, popliteal and mesenteric), oesophagus, stomach, duodenum, colon, urinary bladder, ureter, uterus, nervus ischiadicus, spinal cord, pancreas, tongue, skin, anal sac, circumanal glands, thoracid aorta, abdominal aorta, skeletal muscle, mammary gland, bone marrow, bone, eye with optic nerve, other gross changes. Bone marrow smears were prepared and fixed in methanol. All tissues preserved, except the tongue, skin, anal sac, circumanal glands, teachea popliteal and bones, were processed for wax embedding and 5 μm sections, stained by hematoxylin and eosin, and examined microscopically.

Other examinations:

FAECAL BLOOD CONTENT
- The faeces samples of all dogs were examined for the presence of occult blood during 4 consecutive days pre-dosing and daily during four consecutive days at week 1, 4, 8 and 12 of treatment.

ORGAN FUNCTION TESTS
- A liver-function test (bromosulphophthalein method) was carried out upon all dogs of the top-dose and control group at week 13. Blood samples were taken at one and at thirty minutes after intravenous injection of 12.5 mg BSP/kg body weight. The retention after 30 minutes was calculated from the extinction values at one and at 30 minutes.
- A kidney-function test was conducted upon all dogs at week 13. The phenolsulphophthaleine (PSP) method was used, After a 16 hour period without food 1 mg PSP/kg body weight was intravenously injected. Next a blood sample was collected after 60 minutes and the concentration of PSP in the plasma was measured.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- A few small ulcera of the buccal mucosa were observed in the male dog 7-364 (high-dose group) after 10 days of treatment. A few days later, the dog became ill, still showing ulcera, accompanied
by a swollen mandibular lymph node. The food intake decreased markedly and slight fever occurred. Therefore, 225,000 IU penicillin and 375 mg streptomycin were administered for five consecutive days. The dog recovered within a week but the food intake continued to be too low. In week 7 of the study the same dog had a moderate purulent conjunctivitis. The next few weeks the dog became thin, showed a mucous discharge from eye and nose, and pale to slightly yellow conjunctivae and buccal mucosae. To prevent the dog from severe emaciation the test material was omitted from the diet from week 10 and onward. Within two weeks the dog had clinically recovered.
- Reddening of the gingivae was observed in female dog 7.303 and in male dog 7.301 both of the top-dose group in week 2 only.
- None of the other dogs showed any abnormalities in health condition. Behaviour was not affected in any of the dogs.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean body weights were distinctly lower in dogs of the top-dose group than in the controls during the major part of the study.
- A similar phenomenon, though to a lesser degree, was observed in dogs of the mid-dose group. One dog of this group gained more weight than did any of the dogs used in the present study. Therefore, the resulting differences between the mean values of mid-dose dogs and the controls were relatively small. Body weights of males of the low-dose group were also slightly lower than those of the controls. This was attributed to the relatively low initial weights of this group, since the weight gain figures were comparable with those of the controls. Weight gain in females of the low-dose group was very similar to that of control females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the first two weeks of the test period all dogs received 35 g/food/kg body weight/day. This quantity appeared to be not sufficient for the dogs. Therefore the quantity was increased to 40 g food/kg body weight/day for the remainder part of the test period. All dogs consumed the daily portions of food given, except for male dog 7.364 of the top-dose group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

A small corneal ulcer was seen in male dog 7.368 of the top dose group at the end of the study. Since corneal ulcers do not occur frequently in dogs of this colony, it is possible that the ulcer was induced by the test material.
No treatment-related changes were observed in the other dogs.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The ingestion of 64 mg/kg body weight/day test material resulted in clear decreases in haemoglobin content, packed cell volume and red blood cell count at week 6 and 13. Moreover, the number of lymphocytes was slightly diminished, while the number of neutrophils was elevated. In dogs fed 16 mg/kg body weight/day the haemoglobin content, packed cell volume and red blood cell count were generally also lower than in the controls, though the differences were not always statistically significant. The increased number of thrombocytes at week 6 in dogs fed 16 mg/kg body weight/day was an isolated finding and was considered to be unrelated to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dogs of the top-dose group showed a slight, though consistent, increase in serum urea values. Total protein and albumin values of this group were slightly, though statistically significantly lower
than those of the controls at week 6. The alkaline phosphatase activities of this group were generally lower than those of the controls, though male dog 7.3.64 showed increased values at week 6
and 13. Since increases, rather than decreases are indicative of adverse effects, the decrease of the alkaline phosphatase activities were not considered to be of toxicological importance. Dogs of the mid-dose group showed significantly lower bilirubin values compared with the controls at week 6 only. Since there was no dose-response relationship, no toxicological significance was attached to this finding.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At week 13, the pH values of the urine of dogs given 16 or 64 mg/kg body weight/day were generally slightly higher than those of the controls. All values however were within the normal range. The remainder of the semi-quantitative tests did not reveal indications of treatment-related differences between test and control dogs. The incidence of cells or of other constituents in urine sediment was similar in all groups. The statistically significant increase of the Na content at the low intake level at week 6, and of the K content at the intermediate intake level at week 12 were isolated findings, and considered unrelated to treatment.

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):

Absolute organ weights were generally similar in the different groups. When, however, expressed relative to body weight, the values of the heart, kidneys, liver, brain and lungs turned out to be statistically significantly higher in dogs fed 64 mg/kg body weight/day than in controls. Slightly higher values of relative liver, brain and lung weights were observed also in the mid-dose group, but the differences with the controls were not statistically significant. Dogs fed the test material showed lower mean thymus weights than the controls. The differences with the controls were however, not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At gross examination male dog 7.364 revealed several changes. The kidneys and gall bladder appeared to be enlarged. The bronchial lymph nodes of this dog were enlarged and haemorrhagic. Moreover slightly brown discoloured adipose tissue was found in the mesentery and pericardium of this dog. A similar discoloration of the adipose tissue was found in the mesentery of two females (viz. 7.325 and 7.392) of the top-dose group.
Many dogs had intestinal parasites, most probably Toxocara canis.
In six female dogs a haemorrhagic mucosa of the urinary bladder was found, but this finding was attributed to catheterisation of the bladder.
Other minor gross lesions were about equally distributed between control and test animals or were isolated findings. These findings were, therefore, considered to be of no-toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes were observed in the brown discoloured fat tissues of the pericardium and/or mesentery.
Slight differences in incidence of mononuclear inflammatory cell infiltrates in the liver, were observed between males of control- and top-dose groups. These differences, however, are not considered to represent a toxic effect since they were only slight and since, moreover, similar differences in incidence are quite common in the colony of dogs used.
A focal area of necrosis surrounded by neutrophilic leucocytes slight periportal fibrosis and a focal sub-acute pericarditis were observed only in high-dose animals. Each of the changes, however, occurred only in one dog. Since, moreover, these alterations are rather common in control dogs used in other toxicity studies, they are considered to be fortuitous findings unrelated to treatment.
Some atrophic glomeruli were seen in the kidneys of two dogs of both the top- and mid-dose group. This change, however, is a very common finding in beagles of this colony; moreover, the incidence in both groups was the same. Therefore, this change was not ascribed to the test material.
Slightly increased deposits of brown pigment were found in a lymph node of one male and one female of the top-dose group. Brown pigment dipositions, in various degrees, are rather frequently observed in lymph-nodes of our dogs. It is therefore highly unlikely that the deposition seen in the present study has any toxicological significance.
The other pathological changes observed were about equally distributed amongst the control group and the different test groups or they occurred only in a single animal. Therefore, none of these abnormalities, which are common findings in beagle dogs of the TNO-colony, are ascribed to the feeding of the test material.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

KIDNEY AND LIVER FUNCTION TESTS
- Specific gravity values of urine were similar in all groups. These values were measured in urine collected from dogs which had constant access to drinking water. No specific gravity values are available of urine obtained after an 18-hour period of water deprivation, because it appeared impossible to collect any urine after such a period. Phenol red retention was statistically significantly higher in dogs fed 64 mg/kg body weight/day than in controls. In the same group the bromosulphophthalein retention was also significantly higher than in the controls. However, a large part of this increase in BSP retention was caused by an extremely high value of male dog 7.364. The BSP retention values of the other males of this group were similar to the control values. These values of the females, however, were slightly higher than those of the controls.

FAECAL BLOOD
- During the pre-treatment period several dogs in each of the groups had once or a few times minimal quantities of occult blood in the faeces. After the initiation of the treatment period only one control dog and one of the intermediate dose group showed only once a minimal quantity of occult blood in the faeces.

Details on results:

The present study revealed changes in dogs of the high-dose group, which are attributed to treatment.
The distinct depression in body weight gain in dogs fed 64 mg/kg bw/day test material was not accompanied by a reduced food intake, which indicates an unfavourable effect on food efficiency. Therefore, the depression of weight gain was considered to be a deleterious effect of the test material.
The decrease in haemoglobin content, packed cell volume and red blood cell count in dogs of the high-dose group were ascribed to treatment, since these chances occurred both after 6 and 13 weeks. Moreover, indications of similar changes were observed also, though less pronounced, in dogs of the mid-dose group.
A major organ affected was the kidney of dogs of the top-dose group. This appeared from an impaired kidney function, elevated serum urea values and increases in relative kidney weights. Microscopically, however, no treatment-related changes were observed. Although microscopy of the liver likewise failed to reveal changes attributable to treatment, the results of the BSP function test and the relative liver weight suggested an effect of the test material in the high-dose group.
The increases of the relative weights of the brain and lungs in the top-dose group were probably unrelated to treatment, because there were no indications of treatment-related histopathological changes in these organs. Moreover, it has been shown that in rats increases in the relative weight of the brain and lungs may occur as a result of depression in body weight gain (Feron et al., Fd. Cosmet. Toxicol. 11 (1973) 85 - 94). Since the body weights of top-dose dogs were depressed a similar relationship may be responsible for the increased relative weights of the brain and lungs observed in these dogs. The increased relative weight of the heart in dogs of the top-dose group is considered to be of doubtful toxicological significance since it was not associated with morphological changes in this organ.
Clinical symptoms were restricted to a few dogs of the top-dose group and were severe in one dog. The rapid improvement of the dog after withdrawing the test material from the diet was considered an indication that the test material had induced the symptoms. Corneal ulcers, as observed in one dog of this group may have been induced also by the test material since they are rarely if ever observed in dogs of the colony used. The reddening of the gingivae observed in two other dogs might be due to the irritating properties of the test material on mucous membranes. It is very doubtful whether the brown discoloured adipose tissue in three dogs of the top-dose group is of any toxicological significance, since the phenomenon was not accompanied by any microscopical changes in this tissue.
Although the thymus weights, both absolute and relative, were generally lower in the test groups than in the controls no statistically significant differences occurred amongst the groups. Since similar low thymus weights were found also in the control group and moreover, no noticeable microscopical changes were observed in this organ, it does not seem justified to attach any toxicological significance to the relatively low weights in the test groups.
Dogs fed 16 mg/kg body weight test material only showed relatively low weight gain and slight, and transitory decreases in red blood cells and packed cell volume. Since these changes occurred more markedly in the top-dose group their occurrence in the mid-dose group cannot be disregarded.
On the basis of these results it seems justified to conclude that 4 mg/kg body weight test material was the no-effect level in the present study.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

haematology

ophthalmological examination

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

64 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

Ophthalmoscopic Changes

Animal No. and Sex	Mg/kg bw/day	Changes Observed	At Week(s)
7.321 M	Control	Slight reddening of the conjunctivae	1
7.314 M	Control	Idem	7
7.382 F	Control	Idem	1
7.304 F	Control	Idem	7 and 13
7.386 M	4	Pale conjunctivae	7
7.309 F	4	Hypertrophy of the membrana nictitans	1, 7 and 13
7.349 F	16	Vascular injection in the cornea	1
7.364 M	64	Pale conjunctivae	7
7.368 M	64	Small corneal ulcer	13
7.392 F	64	Slight reddening of the conjunctivae	7

 

Mean Body Weights and Mean Weight Gain

Mg/kg bw/day	Body Weight (in kg) at Week							Weight Gain (kg)
	0	2	4	6	8	10	13	1-13
Males
0	8.1	8.6	9.4	10.2	10.9	12.0	12.9	4.8
4	7.6	8.0	8.9	9.6	10.3	11.1	11.8	4.2
16	7.8	8.3	8.9	9.6	10.3	10.8	11.8	4.0
64	8.0	7.8	8.1	8.6	8.6	9.0	9.9	1.9
Females
0	6.4	6.6	7.1	7.5	8.1	8.4	9.2	2.8
4	6.3	6.3	6.7	7.2	7.8	8.1	8.9	2.6
16	6.3	6.2	6.5	7.0	7.4	7.5	8.3	2.0
64	6.4	6.2	6.4	6.7	7.1	7.4	8.1	1.7

 

 

Concentrations of Test Material Recovered in the Diets

Group (mg/kg bw/day)	Concentration of Test Material (in ppm) in Diet Samples Taken After Storage at Day			Nominal Concentration (ppm) at Day
	0	7	28	1 (2))	43 and 84 (3))
4	101 (89)	97 (95)	95 (93)	113	102
16	437 (96)	411 (104)	370 (93)	454	397
64	1840 (101)	1537 (97)	1602 (101)	1814	1587

 

() in brackets the percentages recovered

2) based on a food intake of 35 g/kg body weight/day

3) based on a food intake of 40 g/kg body weight/day

 

Mean Haematological Indices

Mg/kg bw/day	Clotting Time (sec.)	Hb (m mol/L)	Packed Cell Volume (L/L)	RBC ((x10^12 /)L)	WBC ((x10^9 /)L)	Thrombocytes ((x10^9 /)L)	Differential Count (%)
							Lymph	Neutr	Mono	Eos	Bas
Week 0
0	26	8.1	0.411	5.0	13.2	335	44	53	1	2	0
4	26	8.1	0.406	5.1	14.8	281	40	54	2	5	0
16	26	8.3	0.411	5.1	15.1	323	41	55	1	3	0
64	26	8.0	0.397	5.0	13.1	415	41	56	1	2	0
Week 6
0	30	8.7	0.436	5.6	12.4	274	37	53	2	8	0
4	28	8.6	0.424	5.5	12.7	323	38	55	2	5	0
16	27	8.0	0.404*	5.1*	12.8	368*	33	60	7	1	0
64	29	6.7***	0.330***	4.2***	14.7	324	25*	66*	1	9	0
Week 13
0	25	9.3	0.456	6.2	13.9	270	39	55	1	4	0
4	25	9.3	0.444	6.2	14.0	258	40	55	1	4	0
16	24	9.0	0.436	5.9	16.1	292	41	56	0	3	0
64	24	7.8**	0.371***	5.1**	14.6	329	32	63	1	4	0

Hb = haemoglobin content

RBC = red blood cell count

WBC = white blood cell count

* P<0.05; ** P<0.01; *** P<0.001 according to Wilcoxon test

 

Mean Biochemical Parameters

Mg/kg bw/day	Glucose (m mol/L)	Urea (m mol/L)	GOT (U/L)	GPT (U/L)	AP (U/L)	TP (g/L)	Albumin (g/L)
Week 0
0	7.0	2.2	3.9	4.0	59	60	32
4	6.9	2.6	4.1	4.6	57	57**	31
16	6.8	2.8	4.0	4.1	54	59	32
64	7.0	2.6	3.6	3.3	53	59	32
Week 6
0	6.2	3.4	6.4	8.5	46	54	29
4	6.5	4.0	6.1	8.0	46	54	27
16	6.4	3.8	5.6	7.7	39	53	26
64	5.9	5.4**	5.0	6.6	33***	49**	23**
Week 13
0	6.3	4.5	6.9	8.1	43	60	32
4	6.5	4.5	6.5	7.8	42	62	33
16	6.4	4.5	5.3	6.2	39	60	32
64	5.0	5.6*	5.5	8.8	35	56	31

Urea = urea nitrogen

GOT = glutamic-oxalacetic transaminase

GPT = glutamic-pyruvic transaminase

AP = alkaline phosphatase

TP = total protein

* P < 0. 05; ** P <0.01; *** P < 0.001 according to Wilcoxon

 

Mean Biochemical Parameters Continued

Mg/kg bw/day	Na in Plasma (mg/100 mL)	K in Plasma (mg/100 mL)	Billirubin (Umol/L)	OCT (U/L)	Creatinine (Umol/L)
Week 0
0	350	16.3	1.6	6.7	59
4	351	16.2	1.7	6.9	56
16	350	15.7	1.8	6.4	57
64	352	16.3	1.8	6.5	57
Week 6
0	351	16.9	1.6	4.8	64
4	327	16.2	1.4	4.8	69
16	337	16.3	1.2*	4.6	60
64	328	16.0	1.6	4.4	59
Week 13
0	372	18.0	1.3	7.9	71
4	371	17.3	1.2	8.2	69
16	378	18.0	1.3	7.5	64
64	386	18.8	1.5	8.0	67

OCT = ornithine carbamoyl transferase

*P<0.05 , according to Wilcoxon test

 

Mean Percentages of Globulins

Mg/kg bw/day	Week 0			Week 6			Week 13
	α	β	γ	α	β	γ	α	β	γ
0	49	31	20	51	33	16	51	32	17
4	51	29	20	51	34	15	53	31	17
16	51	29	20	51	34	15	53	32	15
64	49	31	20	53	32	15	53	31	16

 

Kidney and Liver Function Tests

Mg/kg bw/day	Specific Gravity at the End of Week			Phenol Red (µg/100 mL)	BSP Retention (%)
	0	6	13	12	12
0	1.0258	1.0412	1.0263	41	3.0
4	1.0248	1.0355	1.0257	38	-
16	1.0284	1.0403	1.0293	44	-
64	1.0324	1.0352	1.0238	74**	6.2*

BSP = bromosulphophthalein

* P < 0.05; ** P < 0.01 according to Wilcoxon test

 

Mean Na and K Content in Urine

Mg/kg bw/day	Na	K	Na	K	Na	K
	In Urine (mg %)
	Week 0		Week 6		Week 12
0	247	227	432	502	253	209
4	267	244	614*	612	337	203
16	398	227	545	556	303	328*
64	405	246	532	529	238	241

* p < 0.05 according to Wilcoxon test

 

Mean Relative Organ Weights (in g/100 g body weight)

Mg/kg bw/day	Heart	Kidneys	Liver	Spleen	Brain	Testes	Ovaries	Pituitary	Thyroid	Adrenals	Lungs	Thymus	Prostate
0	0.80	0.49	3.52	0.22	0.69	0.14	0.0077	0.00065	0.0084	0.0109	0.87	0.18	0.039
4	0.82	0.49	3.52	0.21	0.67	0.15	0.0077	0.00069	0.0088	0.0106	0.85	0.15	0.020
16	0.81	0.48	3.72	0.20	0.78	0.14	0.0100	0.00067	0.0091	0.0120	0.95	0.14	0.032
64	0.94*	0.59**	4.40***	0.24	0.87*	0.14	0.0091	0.00076	0.0095	0.0113	1.01*	0.12	0.033

* P < 0.05; ** P < 0.01; *** P < 0.001 according to Wilcoxon test

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.

Executive summary:

A sub-chronic (90-day) feeding study with the test material was performed in beagle dogs fed diets containing the test material at levels which provided intakes of 0, 4, 16 or 64 mg/kg body weight/day. The study was similar in design to OECD 409.

Health, condition and behaviour were generally not affected by the test material. One dog of the top-dose group showed ulcera in the buccal mucosa, a purulent conjunctivitis, pale mucous membranes and a poor condition. Gain in body weight was markedly decreased in dogs of the top-dose group and slightly decreased in dogs of the intermediate-dose group. Food consumption was decreased only in one dog of the top-dose group. Haemoglobin content, packed cell volume and red blood cell counts were decreased in the top-dose group. Rather low value occurred in the intermediate-dose group. In the top-dose group there were moderate increases in serum urea values. Phenol-red, and BSP-retention were increased in the top-dose group. The pH of the urine was generally slightly higher in dogs of the two highest dose groups than in controls. On ophthalmoscopical examination no treatment-related changes were observed. A purulent conjunctivitis was seen in one dog of the top-dose group.

The relative weights of the heart, kidneys, and liver were increased in the top-dose group. On gross examination brown discoloured fat tissue in pericard and/or mesenterium was observed in three dogs of the top-dose group. Microscopic examination was essentially negative.

Under the conditions of this study, the NOAEL was 4 mg/kg bw/day.