Prometryn

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Nov 1989 to 30 Jan 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

12 May 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Pesticide Assessment Guidelines, Subdivision F

Version / remarks:

November 1984

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Ministry of Agriculture, Forestry and Fisheries, Japan, 59 NohSan No. 4200

Version / remarks:

28 January 1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Prometryn

EC Number:

230-711-3

EC Name:

Prometryn

Cas Number:

7287-19-6

Molecular formula:

C10H19N5S

IUPAC Name:

6-(methylsulfanyl)-N2,N4-di(propan-2-yl)-1,3,5-triazine-2,4-diamine

Test material form:

solid: particulate/powder

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 7 months
- Weight at study initiation: 8.5 ± 0.8 kg (males) and 7.8 ± 0.5 (females)
- Housing: Dogs were housed in individual stainless steel cages (0.75 x 1.00 x 0.82 m) during the acclimatization and treatment periods, equipped with a metabolism tray for the collection of urine and faece
- Diet: Each animal had free access to approximately 400 grams of fine ground diet, distributed daily, except as noted in "Laboratory investigations".
- Water: Tap water was distributed ad libitum to the animals by automatic stainless steel pipettes.
- Acclimation period: about 6 week.

DETAILS OF FOOD AND WATER QUALITY:
The batches of food used in this study were analysed forhomogeneity, physical quality, nutritive quality, bacteriological contaminants and mycotoxins, organochlorine and organophosphorus pesticides, heavy metals and nitrosamines. Routine bacteriological and chemical analyses of water were made in order to detect possible major contaminants. There was no information available indicating that any non-nutrient substances at a level likely to influence the effect ofthe test substance, were present in the diet or in the water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 / 12
- Air changes (per hr): 10 - 15 volumes per hour without being recycled.

IN-LIFE DATES: From: 21 Sep 1989 To: 07 Feb 1990

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Each week for each group, the test substance was blended in a mortar with a small quantity of the diet. The required concentration was then prepared by direct dilution of each premix with further quantities of untreated diet. Homogeneity was obtained by mixing in a mixer type for 15 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the beginning of this study, the homogeneity of a dietary mixture containing 20 ppm of test substance was checked. The results of these analyses showed a good homogeneity of this preparation which was sampled at 3 different levels. The stability of the same dietary mixture was then checked. The preparation was kept either in closed bags sampled the day of preparation and after 2, 4, 7, 10 and 14 storage days at room temperature or in open feeders sampled the day of preparation and after 24 storage hours in the animal room.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

Group A, low dose: Dietary equivalent to 0.81 and 0.89 mg/kg /day for males and females, respectively.

Doses / concentrations 2

Dose / conc.:

200 ppm

Remarks:

Group B, mid dose: Dietary equivalent to 8.26 and 8.13 mg/kg /day for males and females, respectively.

Doses / concentrations 3

Dose / conc.:

2 000 ppm

Remarks:

Group C, high dose: Dietary equivalent to 70.61 and 82.80 mg/kg /day for males and females, respectively.

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS
All clinical signs were recorded once a day in each animal at the sameapproximate daily time.

MORBIDITY AND MORTALITY
All animals were checked each day at approximately 8.00 a.m. and 4.00 p.m. for illness, moribund animals and death. During weekends and Public Holidays, the final check was carried out around midday

BODY WEIGHT
The weight of each animal was recorded once before the beginning of the treatment, on the first day of treatment and once weekly during treatment and recovery periods.

FOOD CONSUMPTION
The quantity of food consumed by each animal was recorded daily, a week before treatment started and during the treatment period. Food intake per animal was recorded by the difference between the initial and final weights of the food container. Fasting of the animals, when necessary, was commenced at approximately 5.00 p.m., the food consumption being recorded at this time.

ELECTROCARDIOGRAPHY
An electrocardiogram was recorded in all animals during weeks 8 and 13. The standard bipolar limb leads (leads I, II, III) were recorded using a
Mingograph by means of hypodermic needles placed subcutaneously. The heart rate (HR) and the duration of PQ, QRS and QT intervals were measured for each animal on each examination.

OPHTHALMOLOGY
The optic media and the fundus of all the animals were examined before the beginning of treatment, during week 13, 2 to 4 hours after drug administration, with an indirect ophthalmoscope. Prior to examination, the pupils of all animals were dilated using an ophthalmic solution after checking pupillary light and blink reflexes.

LABORATQRY INVESTIGATIONS
Blood samples were taken, approximately 24 hours after food distribution, from the external jugular vein of the overnight fasted animals, without anaesthesia. The samples were collected into tubes containing the appropriate anticoagulant (see below). For urine collection, the animals were deprived of food for an overnight period of about 18 hour
 
HAEMATOLOGY
The following parameters were determined in all animals before the beginning of treatment and on weeks 4, 8 and 13 * The parameters with indexes were determined only on weeks 8 and 13. Parameters:
- Blood collected on complexion: Erythrocytes (RBC), Haemoglobin, Mean Cell Volume (MCV), Packed Cell Volume (PCV), Mean Cell Haemoglobin Concentration, Differential White Cell Count (neutrophils (N), eosinophils (E), basophils (B),  lymphocytes (L),  monocytes (M)). Reticulocyte Count (Retic), Heinz bodies (Heinz-B)
- Blood collected an lithium heparinate: Methaemoglobin (METHB)
- Blood collected on sodium citrate: Quick time (QT), Activated Partial, Thromboplastin Time (APTT), Fibrinogen (FIBRIN)

BLOOD BIOCHEMISTRY
The following parameters were determined in all animals before the beginning of the treatment and on weeks 4, 8 and 13
- Blood collected an lithiua heparinate (Sodium (Na+), Potassium (K+), Chloride(Cl-), Calcium (Ca++), Inorganic Phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), bilirubin(T0T.BIL), Total proteins (PROT), Cholesterol (CH0L), Triglycerides (TRIG), Alkaline phosptasetase(ALP), Aspartate amino-transferase(ASAT), Alanine amino- IU/1transferase (ALAT), Gamma glutamyltransferase (GGT)
- Blood Gollftcted without any anticoagulant: Protein electrophoresis: albumin (ALB), alpha-1 globulins (A1-GL0B), alpha-2 globulins (A2-GL0B), beta-globulins (B-GL0B),  gamma-globulins (G-GL0B),  albumin/globulins ratio (A/G).

URINALYSIS
The following parameters were determined in all animals before the beginning of the treatment and on week 13:  volume (VOLUME, ml), appearance (APP), pH (PH), specific gravity (SP.GRAV), urobilinogen (UROB, \]% * Ehrlich unit/100 ml), proteins (PROT), glucose (GLUC), ketones (CETO), bilirubin (BILI), blood (BLOOD), nitrites (NITR) using N-Multistix SG test strips,  microscopy of sediment after centrifugation: investigation for leucocytes (WBC), erythrocytes (RBC), hyaline (HYAL) and granular (GRAN) cylinders, magnesium ammonium phosphate (AMM.PH.), calcium phosphate (CAL.PH) and calcium oxalate (CAL.OX) crystals, epithelial (EPITH.), bladder (BLAD) and kidney (KIDN) cells.

Sacrifice and pathology:

PATHOLOGY / SACRIFICE
On completion of the last treatment and after about 16 hours fasting, all surviving animals were anaesthetized by intravenous injection of thiopental sodium and sacrificed by exsanguination. Moribund animals were sacrificed in the same way.

ORGAN WEIGHTS
In all animals, the following organs were weighed wet as soon as possible after dissection: adrenals, kidneys, testes, brain, thymus, heart, ovaries, thyroids, liver, and parathyroids. Paired organs were weighed separately. The animals were weighed before necropsy.

MACROSCOPIC EXAMINATION
A complete macroscopic examination was performed on all the animals.
All macroscopic observations were recorded individually.

MICROSCOPIC EXAMINATION
All the tissues required for the microscopic examination were embedded in paraplast. sectioned at approximately 4 microns in thickness and stained with hemalum-eosin. Microscopic examination was performed on all macroscopic lesions and tissues as listed above for all animals.

PRESERVATION OF TISSUES
In all animals, sample of the macroscopic lesions and the following tissues were preserved in 10% buffered formalin, except the eyes and pituitary gland which were fixed in fonnol-sublimate. See Table 1 in the section "Any other information on materials and methods incl. tables".

Statistics:

The following sequence was used for the statistical analysis of body weight, food consumption, electrocardiography, haematology, blood biochemistry, urinalysis and organ weight data:
The normality of the distribution of the values in each group was checked by Kolmogorov-Smimov’s test (1948).
If the distribution was normal, the homogeneity of variances between the groups was assessed by Bartlett’s test (1937) (more than 2 groups) or Fisher’s test (1934) (2 groups). If no significant heterogeneity of the variances was established, the comparison between treated and control groups was performed by Dunnett’s test (1955).
If the variances were heterogeneous, the comparison between treated and control groups was performed by Dunn’s test (1964) (more than 2 groups) or by Mann Whitney’s test (1947) (2 groups). If the distribution of values in the groups was not normal, the analysis was repeated after logarithmic transformation of the values (except for organ weights).
If this logarithmic transformation failed to normalise the distribution of the values, comparison of treated and control groups was performed by Dunn’s test using original values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed throughout the study.

Mortality:

no mortality observed

Description (incidence):

No mortality was noted during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight of the animals was normal throughout the study, and comparable in treated and control groups. However, one control male lost 1 kg between week 1 and week 14. If this animal is not taken in account, it could be considered that the body weight gain of the males of the 2000 ppm group was very slightly reduced when compared with the animals of the other groups. In this case, this change could be related to the slight decrease in food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was normal and comparable between the males and females of the 20 and 200 ppm groups, the females of the 2000 ppm group and the control animals. A moderate decrease was observed during the first week of treatment in the males receiving 2000 ppm compared with the pretreatment values and the other animals (about 30% compared with the pretreatment values). Thereafter, the food consumption remained very slightly inferior until the end of the study (about 3% compared with the pretreatment values).
The mean achieved intake of test substance was 0.81, 8.26 and 70.61 mg/kg/ day for the treated males, and 0.89, 8.13 and 82.80 mg/kg/day for the treated females. Except for the males of the high dose group, these values coresponded to the factor 10 chosen for the concentrations. The achieved intake of test substance could be considered as practically stable ever though slight body weight variations were observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few animals showed a hypopigmentation of the fundus. These variations from normal are not uncommon in dog eye examinations (Bellhom, 1981; Rubin, 1970). These variations were, moreover, observed at predose and without change in week 13, and consequently were not related to the treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On week 8, a slight decrease in mean haemoglobin, mean cell haemoglobin and mean cell haemoglobin concentration was found in all male groups including the control group, when compared to the predosing values. On week 13, the values of mean haemoglobin in the males of the 2000 ppm group were lower than the controls, but these were comparable to those observed on week 8. These were considered to be of no toxicological significance, since there was no progressive decrease in these parameters, and the same changes were also found in the controls on week 8. Moreover, the individual values were within the normal range of our background data (haemoglobin: 12.1 - 16.8 g/dL). A slight increase in platelets was observed in the males of the 2000 ppm group on weeks 8 and 13, when compared to the pretreatment values. However, an increase in the platelet count was also found in the control animals on week 13, when compared with their values on week 8 and pretreatment period. Therefore, this was not considered to be toxicologically meaningful. A slight decrease in activated partial thromboplastin time was noted in the males of the 2000 ppm group on weeks 4, 8 and 13. However, this was minor, and the individual values were, in almost all animals, within the normal range of our background data. Therefore it was considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slight decrease in urea was observed on weeks 4, 8 and 13 in the males of the 2000 ppm group. However, this was due to slightly high values in some controls, and the individual values in the males of the 2000 ppm group were higher to one lower normal limit (2.2 mmol/L); therefore, it was not considered to be treatment-related. No abormalities were observed in the other blood biochemical parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The results of the urinalysis were normal.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Slight increase in mean absolute and/or relative weights were found in some organs (kidneys, liver, thyroids, heart and ovaries) of treated animals. Except for the increase in liver absolute and relative weights in the 2000 ppm grows, these increases were minimal and the individual values were comparable between the control and treated animals. Furthermore, the Increase in liver weight at the dose level of 2000 ppm was the contribution of a moderate increase in liver weight one of the animals. The increase in liver weight in the 2000 ppm group was considered to be a treatment-related effect and could be related to the hepatocyte hypertrophy seen in this animal. In the absence of any relevant histopathological findings, the variations in weight of the other organs were considered to be of no toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The few macroscopic changes noted at necropsy were commonly recorded findings in the laboratory dogs. Therefore they were considered to be of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Focal chronic hepatic cell necrosis associated with moderate fibroplasia were found in two animals of the 2000 ppm group. In one of the above-mentioned animals; moderate hepatic cell hypertrophy was observed. This was in line with the increase in liver weight noted in this animal. In addition, Kupffer's cell laden pigments were noted in one male from the same group. The hepatocyte hypertrophy, necrosis and fibroplasia together with the increase in liver weight were considered to be treatment-related. The Kupffer's cell laden pigments are occasionally spontaneously observed in the untreated dog. Accordingly, the relationship of these hepatic changes to the treatment was considered unlikely.
The few microscopic changes noted in the other organs were commonly recorded findings in the laboratory dogs. In addition, their incidence, severity and morphological characters were comparable in all groups including the control. Therefore they were considered to be of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

ELECTROCARDIOGRAPHY
The values of heart rate and PQ, QRS and QT intervals were similar in treated and control animals and remained within the normal range for the species. No abnormal deflection was seen on the electrocardiograms of the animals.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

200 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Dietary equivalent to about 8.3 and 8.1 mg/kg bw/d for males and females, respectively

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

VERIFICATION OF FORMULATION STABILITY

The homogeneity and the stability of dietary mixtures were checked in a previous study  for test substance concentrations ranging from 100 to 5000 ppm, and the results were satisfactory. The results of the analyses showed a good stability for the 20 ppm dietary mixture under the above-mentioned conditions. On weeks 1, 4, 8 and 12, each preparation (control group included) was checked for achieved  oncentration of the test substance. The results of the analyses revealed a satisfactory correlation between obtained and theoretical values.

Table 1. Body weight (mean values - kg) Male

Concent. ppm week		0	20	200	2000
-1	M (3) (SD) n	8.4 0.86 4	8.3 1.12 4	8.3 0.89 4	8.3 0.83 4
1	M (3) (SD) n	8.5 0.73 4	8.5 1.18 4	8.6 0.71 4	8.6 0.73 4
2	M (3) (SD) n	8.7 0.69 4	8.5 1.19 4	8.8 0.79 4	8.6 0.72 4
3	M (3) (SD) n	8.6 0.64 4	8.5 1.33 4	8.9 0.76 4	8.6 0.58 4
4	M (3) (SD) n	8.7 0.73 4	8.6 1.29 4	8.9 0.84 4	8.6 0.67 4
5	M (3) (SD) n)	8.6 0.69 4	8.7 1.32 4	9.1 0.92 4	8.6 0.68 4
6	M (3) (SD) n	8.7 0.79 4	8.9 1.30 4	9.2 0.95 4	8.8 0.71 4
7	M (3) (SD) n	8.7 0.62 4	8.8 1.32 4	9.0 0.99 4	8.8 0.54 4
8	M (3) (SD) n	8.8 0.64 4	8.8 1.27 4	9.1 0.91 4	8.8 0.70 4
9	M (3) (SD) n	9.2 0.74 4	9.2 1.40 4	9.1 0.96 4	9.1 0.59 4
10	M (3) (SD) n	8.9 0.59 4	9.3 1.26 4	9.4 0.98 4	9.2 0.72 4
11	M (3) (SD) n	8.8 0.75 4	9.2 1.28 4	9.5 0.91 4	9.1 0.77 4
12	M (3) (SD) n	8.9 0.65 4	9.3 1.26 4	9.5 0.93 4	9.2 0.75 4
13	M (3) (SD) n	8.9 0.70 4	9.5 1.24 4	9.7 0.73 4	9.2 0.87 4
14	M (3) (SD) n	8.9 0.75 4	9.5 1.16 4	9.6 0.88 4	9.1 0.59 4

Significance of the difference between treated and control groups:

*  P<0.05

** P<0.01

(1) Dunnett test

(2) Mann-Whitney test

(3) Dunn test

Sample distribution-relative tests

(B) Barlett test P<0.01

(F) Fisher test P<0.01

(K) Kolmogorov mirnov test P<0.01

- Statistics excluded group

Applicant's summary and conclusion

Conclusions:

In the current study, a daily administration of the test substance by oral route to the Beagle dog for a period of 13 weeks showed a dose level of 200 ppm (corresponding to about 8 mg/kg/day) to be the no-adverse effect level under the conditions of this study. 

Executive summary:

A study complying to GLP and OECD guideline 409 investigated the toxic effects of the test substance administered by oral route (diet mixture) for a period of 13 weeks to the Beagle dog. Thirty-two animals (16 males and 16 females) were randomly allocated to one control and 3 treated groups each containing 4 males and 4 females. The test substance was administered daily by dietary admixture at dose levels of 20, 200 and 2000 ppm for 13 weeks, corresponding to doses of about 0.8, 8 and 80 mg/kg bw/day. Examinations performed daily included clinical examination of the animals and a calculation of food consumption. Body weights were recorded before the beginning of treatment and once a week during the study. An electrocardiographic examination was performed on weeks 8 and 13. An ophthalmologic examination was performed before the start of treatment and on week 13. Haematological and blood biochemical examinations were performed before the study begin and on weeks 4, 8 and 13. Urinalysis was performed before the study start and on week 13. At the end of the treatment, the animals were sacrificed. Organ weights were recorded. The organs were submitted to a full macroscopic examination and tissue specimens were examined microscopically. 

Clinical signs: No clinical signs were observed throughout the study. Mortality: No mortality occurred during the study. Body weight: Taking into consideration that one control male lost 1 kg throughout the treatment period, it could be noted that the body weight gain of the males treated at 2000 ppm was slightly reduced. The daily administration of the test substance by oral route (admixed to the diet) to the Beagle dog for a period of 13 weeks was well-tolerated without evidence of systemic toxicity at the dose levels of 20 and 200 ppm. At the dose level of 2000 ppm, a slight decrease in both food consumption and body weight gain was noted in the males. Moreover, a moderate increase in liver weight related to a hepatocyte hypertrophy in combination with hepatic cell necrosis and fibroplasia was found in few animals receiving 2000 ppm. No other signs of adverse effects following repeated oral exposure to the substance occurred in this study.

In conclusion, the dose level of 200 ppm (corresponding to about 8 mg/kg/day) was the no-adverse effect level under the conditions of this study.