Ametryn

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in compliance with guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ametryn

EC Number:

212-634-7

EC Name:

Ametryn

Cas Number:

834-12-8

Molecular formula:

C9H17N5S

IUPAC Name:

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

Details on test material:

Name: Ametryn technical
CAS No. : 834-12-8
Manufactured and : Oxon Italia SpA
Lot No. : S349
Date of manufacture : October, 1996
Date of expiry : October, 1998
Declared purity : 96% (960 g/kg)
Physical appearance : Solid, white powder
Solubility : Soluble in water at 185 mg/L and also soluble in acetone at 500 g/L

Test animals

Species:

rat

Strain:

other: hsdcpb: WU rats conventionally bred

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at the start of the experiment : 5 - 6 weeks

Mean body weights (g)
± SD at the start of treatment :
Males Females
G1: 92 ± 8.6g 87 ±8.5g
G2: 93 ± 9.7g 88 ± 9.5g
G3 93 ± 9.7g 87 ± 9.9g
G4: 94 ± 9.3g 87 ± 8.8g
G5: 92 ±11.7g 87 ± 6.5g

Identification : Rat accession number, cage card and corresponding picric acid body markings.

Acclimatization : Five days under experimental conditions after veterinary examination.

Animal selection : Before initiation of the treatment, the rats were stratified according to their body weights and assigned to 5 groups of twelve rats each (6 males and 6 females).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test compound was mixed in the laboratory animal feed at concentrations of 750 (G2), 1500 (G3), 3000 (G4) and 6000 (G5) ppm and fed to 4 groups of rats. A concurrent control group (G1) received laboratory animal feed without the test compound admixture.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test compound (Ametryn) extracted from the experimental rats/mice feed with acetone was estimated by means of GLC with Nitrogen Phosphorus Detector (NPD). The achieved concentrations were within the permissible 10% range of nominal concentrations.

The identity of the test compound was confirmed prior to the start of the study. The stability of the test compound in experimental feed was analysed prior to the start of the study. Dietary concentrations of the test compound (Active Ingredient) were determined twice during the study period. Homogeneity in the experimental feed was analysed at the first batch of feed preparation. Three samples from top, middle and bottom layers of the fortified feed from all the tested doses were used for determining homogeneity.

Duration of treatment / exposure:

28 days

Frequency of treatment:

All test groups received the experimental feed specifically prepared for each group ad libitum. The treatment was given 7 days a week for 4 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 ppm (G1), 750 ppm (G2), 1500 ppm (G3), 3000 ppm (G4), 6000 ppm (G5)
Basis:
nominal in diet

No. of animals per sex per dose:

12 rats (6 males, 6 female)

Control animals:

other: yes, a concurrent control group (G1) received laboratory animal feed without the test compound admixture.

Details on study design:

G1: Vehicle control
G2: Low dose
G3: Mid dose
G4: High Intermediate
G5: High dose

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

Veterinary examination was done before grouping and at the end of each week of the experimental schedule as per the standard operating procedure and the findings were recorded.

Ophthalmological examination of all animals was done at the start of the experiment and at the end of the treatment prior to sacrifice with ophthalmoscope. Mydriasis was produced before examination using 1% Tropicamide solution (Akhil Farma Pvt. Ltd., Muttangi 502300, INDIA).

Clinical signs and pre-terminal deaths: Rats were observed for clinical signs and pre-terminal deaths once daily.

Sacrifice and pathology:

Gross necropsy: The animals at term were fasted overnight (water provided) and exanguinated under ether anaesthesia and were subjected to detailed necropsy by a pathologist.

Organ weights: The following organs were collected and weighed: adrenals, gonads, liver, spleen, kidneys, epididymides, thymus, brain and heart The organ weight ratios as percentages of body weights were determined and recorded.

Other examinations:

Individual body weights were recorded at the beginning and at the end of each week of the study.

The weekly feed consumption was calculated by adding the feed consumed in I (4 days) and II (3 days) and the total divided by the number of rats per cage to determine the feed intake/rat/week. The weekly feed consumption (g/rat) was divided by the number of days (7) and expressed as (g/rat/day). This was repeated during each week of the experimental period.

Statistics:

Using specific computer programmes, body weights, net body weight gains, feed consumption, organ weight data and organ weight ratio values were compared by Bartletts test for homogeneity of intra group variances. When the variances proved to be heterogeneous, the data were transformed using appropriate transformation. The data with homogeneous intra group variances were subjected to one-way analysis of variance (ANOVA - Snedecor and Cochran, 1980).
Following ANOVA, when F value was significant, Dunnetts pairwise comparison (Scheffe, 1953) of means of treated groups with the control mean was done individually. Following a significant difference of a test group with the control group, the dose response correlation was estimated by including the control and all the treated groups employing t test.
All analyses and comparisons were evaluated at the 5% level. Throughout this report statistically significant differences, indicated by the aforementioned tests were designated by the superscripts as stated below:

+/- : Significantly higher (+)/lower (-) than the control group
d : Significant dose correlation.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Veterinary and ophthalmological examinations, clinical signs and pre4erminal deaths:
Emaciated condition was observed in three males and all the females and an isolated incidence of perineum wet was observed in one female and 3/6 males and 6/6 females died pre-terminally (one male on day 7 and one female on day 9 were sacrificed moribund due to emaciation between the 5th and 10th day of the treatment period) in which 2 males and 3 females were autolysed at the 6000 ppm dose.
The pre-terminal deaths at the high dose could be due to the unpalatable nature of the test compound which resulted in weakness and emaciation brought about by inanition.

Body weights and cumulative net weight gains:
Individual body weights were recorded at the beginning and at the end of each week of the experiment.
At the 1500 ppm dose, the body weights were significantly lower at weeks 1 and 3 and cumulative net weight gains were lower throughout the treatment period in males and at weeks 1 and 2 in females.
At the 3000 ppm dose, the body weights and cumulative net weights gains were significantly lower throughout the treatment period in both the sexes.
At the 6000 ppm dose, the body weights and cumulative net body weight gains were significantly lower throughout the treatment period in males and at week 1 of the females.
The decrease observed in the mean weekly body weights and cumulative net body weight gains in both sexes were dose-related.
The decrease observed could be partly due to less feed consumption and the general/mild toxicity of the test compound.

Feed intake:
- Males: At the 3000 and 6000 ppm doses, the feed consumption was significantly lower throughout the treatment period. The decrease observed was dose- related for 2nd, 3rd and 4th week of the experimental period.
- Females: At the 3000 and 6000 ppm doses, the feed consumption was significantly lower for the first 2 weeks of the treatment period. -
The decrease observed could be due to the unpalatability of the test compound at the respective dose levels.

Efficiency of feed utilization: The feed conversion efficiency was not altered by the treatment.

Terminal fasting body weights, organ weights and organ weight ratios:
The terminal fasting body weights were significantly lower at the 3000 ppm and 6000 ppm doses in males and at the 3000 ppm dose in females. The decrease in the fasting body weights in males was dose-related.
In males, the absolute weight of adrenals, kidneys, liver, heart, brain and spleen at the 3000 ppm and 6000 ppm doses, testes and epididymes at the 6000 ppm dose, thymus at all the doses were significantly lower. The relative weight of kidneys, liver, heart and brain at the 3000 and 6000 ppm doses, adrenals at the 6000 ppm dose, testes at the 3000 ppm dose and liver at the 1500 ppm dose were significantly higher The relative weight of epididymides at the 6000 ppm dose, thymus at the 750 and 6000 ppm doses were significantly lower. The changes observed were dose-related excepting for changes in the relative weight of thymus and testes.
In females, the absolute weight of adrenals, ovaries, thymus and spleen at 1500 and 3000 ppm doses, heart at the 750 and 3000 ppm doses, brain at 3000 ppm dose were significantly lower. The absolute weight of liver at the 1500 and 3000 ppm doses were significantly higher. The relative weights of adrenals, ovaries and spleen at the 1500 ppm dose and adrenals at the 3000 ppm dose were significantly lower. The relative weight of liver was significantly higher at the 1500 and 3000 ppm doses.
The significant reduction in the fasting body weights were in turn reflected in the changes in the organ weights.

Necropsy:
- External pathology: Emaciated condition was observed in three males and all the females at the 6000 ppm dose.
- Visceral organ pathology: No gross pathological changes were observed in any of the doses tested. Two females were partially cannibalised at the 6000 ppm dose.

Effect levels

Dose descriptor:

NOEL

Effect level:

750 ppm

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified