N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

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:

July - October 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is carried out to GLP standards and is based on the OECD 407 test. A NOEL is calculated based on changes in body weight.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

This study has been performed in compliance with the Procedures and Principles of Good Laboratory Pratice (March 1986) issued by the Swiss Federal Department of the Interior and the Intercantonal Office for the Control of Medicaments.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Name of test material (as cited in study report): CA 2343A (Intermediate of CGA 293343)
- Physical state: fine white powder
- Analytical purity: 96.7 %
- Lot/batch No.: P.503005
- Expiration date of the lot/batch: December 1996
- Storage condition of test material: 0-5 °C

Test animals

Species:

rat

Strain:

other: Tif:RAIf (SPF), hybrids of RII/1 x RII/2 (Sprague-Dwaley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY Limited, Stein, Switzerland
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: 161.1 - 214.5 g in males and 139.1 - 168.1 g in females
- Fasting period before study: no
- Housing: individual housing in Macrolon cages type 3 (area: 900 cm²) with wire mesh tops and standardized granulated soft wood bedding (Societé Parisienne des Scuires Pantin)
- Diet: ad libitum; pelleted, certified standard diet (Nafag No. 8900 FOR GLP); contamitant levels were within the acceptable limits and were not considered to have affected the conduct of the study or the interpretation of the data
- Water: ad libitum; contamitant levels were within the acceptable limits and were not considered to have affected the conduct of the study or the interpretation of the data
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): the approx. amount of diet needed for the whole treatment period was prepared in one batch; the pellets were subsequently airdried and then stored in a deep freezer (approx. -20 °C); food portions for one week feeding were thawed at weekly intervals
- Mixing appropriate amounts with (Type of food): pulverized diet
- Storage temperature of food: approx. -20 °C

Dose levels: 0, 50, 200, 1000, 5000 ppm results in 4.2, 16.3, 87.2, 413.2 (males) and 4.0, 15.7, 87.9, 375.8 mg/kg bw/d (females)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the beginning of the study, food samples containing the test article (batch no. P.503005) at concentrations 25, 250 and 2500 ppm were dispatched to Crop Protection Division (Ciba-Geigy Ltd.) for determination of content, homogeneity and stability.

Determination of concentrations in diet with HPLC:
Appropriate portions of diet, 10 g, were extracted with phosphate buffer pH 7 and methanol. Aliquots of the extracts were appropriately diluted with 0.02M phosphoric acid and the test article was determined by HPLC using a two column switching system and UV detection.


Acceptability criteria:
Content: An analytical result of test article content in the range between 75 % and 125 % is considered acceptable.
Homogeneity: A batch of diet/test article mixture is considered homogeneous, when of 3 samples (A,B,C) taken at beginning, middle and end of pelleting process no sample differs from the mean value by more than 15 %.
Stability: The test article in diet is considered stable, when the content does not decline more than 10 % during the dosing period.

Stability:
The results indicated stability of the test article under 5 weeks freezer conditions (approx. – 20 °C) and after storage for 1 week at room temperature.

Homogeneity:
Homogeneity of the test article distribution in pelleted food was also analyzed in samples from a pretest food preparation. The results showed a homogeneous distribution of the test article in the diet.

Concentrations:
The results of these analyses showed that the contents of CA 2343A in the diet were in agreement with the nominal concentrations. The analyses revealed concentrations of 96 % - 108 % of the nominal values, resulting in mean concentrations of 97 % - 105 %.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 ppm (control)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
200 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Doses / concentrations 5

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5-10 animals per sex per dose (see also Table 1 below);
in each dose group 5 animals per sex and group wre sacrificed at the end of the treatment period (experimental group I);
5 animals per sex in the control and high dose groups were kept on control diet for a consecutive 4-week recovery period before sacrifice (experimental group II)

Control animals:

yes, concurrent no treatment

Details on study design:

DOSE SELECTION RATIONALE:
- dose levels were based on the results of the following previously conducted study: Project no. 962038, Short/Long-term Toxicology, Ciba-Geigy Limited, Stein, Acute oral toxicity inrats (Gavage): LD50 in both sexes > 1000 mg/kg, < 2000 mg/kg
- the following dose levels were selected :
* 50 ppm: expected to result in a daily intake of about 5 mg/kg body weight and to cause no observable adverse effects
* 200 ppm: expected to result in a daily intake of about 20 mg/kg body weight and to cause no or minimal adverse effects
* 1000 ppm: expected to result in a daily intake of about 100 mg/kg body weight and to cause minimal adverse effects
* 5000 ppm: expected to result in a daily intake of about 500 mg/kg body weight and to cause observable adverse effects, but no or few fatalities to permit a meaningful evaluation of the study


TREATMENT:
- over a period of 4 weeks on a main group (experimental group I)
- animals were sacrificed at the end of the treatment period

RECOVERY:
- after treatment period, animals of experimental group II were kept on control diet for a consecutive recovery phase of 4 weeks before sacrifice

Positive control:

None

Examinations

Observations and examinations performed and frequency:

IN-LIFE OBSERVATIONS
- time schedule: daily
- observations were recorded at least weekly
- Detailed clinical observations were performed during pretest and once weekly thereafter at about the same time each day
- Animals were observed in their home cage, during handling and in openfield
- Observations also included test for sensorimotor functions


MORTALITY
- all animals were checked daily (a.m. and p.m.)

BODY WEIGHT: Yes
- Time schedule for examinations: all animals were recorded individually at weekly (midweek) weighing sessions
- First weights were recorded during the acclimatization period


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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was recorded weekly and was calculated for periods of one week


FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- FOBs were conducted at weeks 4 and 8 (recovery groups only), at about the same time each day
- Animals were observed in their home cage, during handling and in openfield
- Observations covered the functional domains of CNS activity, CNS excitation, sensorimotor, autonomic, and physiological functions
- Neurological examinations included test for sensorimotor functions (approach, touch, vision, audition, pain, vestibular), autonomic functions (papillary reflex, body temperature), sensorimotor coordination (grip strength, landing foot splay)


MOTOR ACTIVITY
- assessment with automated openfield device (DIGISCAN Animal Activity Monitor; Omnitech Electronics Inc., Columbus, OH, USA) which allows measurements of increased and decreased motor activity
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (all animals) and additionally at the end of the recovery period (control and high dose animals)
- Anaesthetic used for blood collection: Yes (Ether)
- Anticoagulants: EDTA
- Animals fasted: Yes (overnight)
- How many animals: all animals at the end of the treatment period and additionally control and high dose animals at the end of the recovery period
- Examined parameters: red blood cell parameters (Erythrocyte count, Haemoglobin; Hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width); white blood cell parameters (leukocyte count, differntial leukocyte count: neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells); blood platelets (thrombocyte count); prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (all animals) and additionally at the end of the recovery period (control and high dose animals)
- Anaesthetic used for blood collection: Yes (Ether)
- Anticoagulants: heparin (blood chemistry), 3.8 % sodium citrate (coagulation testing)
- Animals fasted: Yes (overnight)
- How many animals: all animals at the end of the treatment period and additionally control and high dose animals at the end of the recovery period
- Examined parameters: Glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, cholesterol, triglycerides, sodium, potassium, calcium, chloride, phosphorous (inorganic), aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transpeptidase


URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Examined Parameters: urine volume, relative density, urine color, pH-value, protein, glucose, ketones, urobilinogen, bilirubin, erythrocytes


MACROSCOPICAL EXAMINATIONS
- at the end of the treatment period all controls and treated animals of experimental group I were exsanguinated under ether anaesthesia and subjected to detailed necropsy
- at the end of the recovery period all controls and treated animals of experimental group II were also exsanguinated under ether anaesthesia and subjected to detailed necropsy
- at necropsy the following weights were recorded from all animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, epidymides, spleen, thyroid
- the following organs and tissues were preserved in neutral buffered 4 % formalin: skin, mammary area, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle with peripheral nerve (scatic nerve), trachea, lung, heart, aorta, submandibular salvary gland (both), liver, pancreas, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, brain (medulla, pons, cerebral and cerebellar cortex), spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), zymbal gland (both), muzzle, tongue, any tissue with gross lesions


MICROSCOPICAL EXAMINATIONS
- after the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to microscopical examination: spleen, mesenteric lymph node, axillary lymph node, femur with joint (including bone marrow), trachea, lung, heart, liver, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), Peyer’s patches (small and large intestine), kidney (both), urinary bladder, testis (both), epididymis (both), prostate, uterus, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve (sciatic nerve), brain (medulla, pons, cerebral and cerebellar cortex), spinal cord, any organ with gross lesions

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see "Details on results")
HISTOPATHOLOGY: Yes (see also "Details on results")

Statistics:

For each time point and parameter a univariate statistical analysis was performed. Nonparametric methods were applied to allow for non normal as well as normal data distribution.
Each treatment group was compared to the control group either by Lepage’s or by Wilcoxon’s two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives. The Lepage test is a combination of Wilcoxon and Ansari-Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheer test is sensitive to monotone dose-related effects.
At each time point FOB data and motor activity data (session totals named as ‘area under the curve’) of treated groups were compared to the control group using multiple t test. Raw and multiplicity-adjusted p-values based on bootstrap resampling were given.

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:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS
One female of the high dose group (5000 ppm) showed hunched posture from day 13 until day 38.
Other clinical signs observed were hair loss and skin lesions. However, their distribution was considered incidental and therefore, not related to treatment.

MORTALITY
There was no mortality during this study.

BODY WEIGHT
Depressed body weight development in high dose males and females (5000 ppm) during the whole treatment period resulted in mean body weights 19 % and 14 %, respectively, below the control means at week 4.
The mean body weight gain for males and females during the treatment period was depressed by 42 % and 47 %, respectively. Therefore, treatment at 5000 ppm clearly exceeded the MD level. During the treatment-free period high dose males persisted on a depressed level whereas in females complete recovery was noted.

FOOD CONSUMPTION
The mean food intakes in high dose males and females (5000 ppm) were markedly depressed during the first treatment week and, to a lesser extent, also during weeks 2 to 4. The overall mean intake was depressed by 17 % and 15 % in males and females, respectively. In group 4 (1000 ppm) the mean intake at week 1 was 8 % and 6 % lower in males and females, respectively. However, the overall intake (total of weeks 1 to 4) was comparable with the respective control values.

FOOD CONSUMPTION RATIOS
The mean food consumption ratios in the high dose group (5000 ppm) were markedly depressed in males (-37 %) and females (-35 %) during the first treatment week but showed a trend to normalization towards the end of the treatment period as well as during recovery. Other changes were of insufficient magnitude to be of toxicological relevance.

WATER CONSUMPTION
The overall water intake was considered increased in the high dose group (5000 ppm). Males and females consumed 16 % and 14 % more than the respective control groups.

FUNCTIONAL OBSERVATIONAL BATTERY
Throughout the entire study neither detailed clinical observations nor functional measurements performed at week 4 and 8 did reveal any change of toxicological relevance.

MOTOR ACTIVITY
At week 4 and 8 motor activity measurements, no significant changes were observed in any of the parameters assessed.

HAEMATOLOGY
At the end of treatment, lower haemoglobin concentrations, hematocrit and minimally lower values of MCH were noted in male and female animals of group 5 (5000 ppm). Additionally, male animals had higher HDW, and females showed lower MCV. Taking together, These results are reflecting a reversible tendency to hypochtomia, anisochromia and microcytosis of red blood cells.
Other changes to haematology parameters were not considered treatment-related.

BLOOD CHEMISTRY
At the end of treatment lower plasma protein levels were noted in animals of group 5 (5000 ppm). In males it was caused by a reversible decrease of plasma globulins, resulting in higher albumin to globulin ratios. Female animals showed decreased values of plasma albumin and globulins, whereby the changes of albumin slightly persisted after the recovery period. The following changes noted at the end of treatment were fully reversible after the recovery period: Animals of group 5 had lower plams glucose, higher plams bilirubin, higher plasma cholesterol and slightly higher plasma triglyceride levels. Additionally, higher activities of aspartate aminotransferase, alanine aminotransferase and gamm-glutamyl transpeptidase were observed in males of group 5.
Other changes to bloodchemistry parameters were not considered treatment-related.

URINALYSIS
At treatment end animals of group 5 (5000 ppm) excreted a slightly larger amount of urine with lower relative density. The changes were reversible within the recovery period.
There was no evidence that treatment with the test article had affected other urine parameters investigated.

ORGAN WEIGHTS AND RATIOS
At the end of the treatment period the mean carcass weights were depressed in high dose males (-20%) and females (-17%), when compared with the control groups. Consequently, the mean organ weights in males (except for brain, testis, thyroid gland, and epidermis) and in females were lower than in the respective control groups, and the organ to body weight ratios were increased (except for female thymus and thyroid gland, which were lower than control values).
At the end of the recovery period the mean carcass weight of high dose males was still depressed by 11 %. The absolute mean weights for liver (-12 %), kidney (-10 %), and spleen (-14 %) were still lower than control means.
Other differences which attained a level of statistical significance were within the expected range and, therefore, not considered of toxicological relevance.

MACROSCOPICAL FINDINGS
There were no treatment-related macroscopical changes observed at necropsy.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

200 ppm

Sex:

male/female

Basis for effect level:

other: overall effects; 200 ppm correspond to a daily intake of approx. 16 mg/kg body weight.

2

Dose descriptor:

LOAEL

Effect level:

1 000 ppm

Sex:

male/female

Basis for effect level:

other: slight hepatotropic effects in both sexes. Slight nephrotropic effects in males typical for an alpha-2-microglobulin accumulation. In some males slight hypocellulrity of the bone marrow . Reversibility was incomplete for hepatotropic effects.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

TEST ARTICLE INTAKE

Mean test article intake values were calculated based on the food consumption ratios. They were corrected for actual amount of test article in the diet according to the results of chemical analysis (analytical concentration as % of nominal concentration).

The calculated mean daily intake of CA 2343 A, based on the nominal concentrations in the diet was 4.36, 16.8, 83.1 and 401 mg/kg body weights in males, and 4.16, 16.2, 83.7 and 365 mg/kg body weight in females.

Corrected for the analytically determined test article concentrations in the diet, the mean daily intake of CA 2343 A was 4.23, 16.3, 87.2 and 413.2 mg/kg body weight in males, and 4.04, 15.7, 87.9 and 375.8 mg/kg body weight in females.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, treatment with 5000 ppm CA 2343 A exceeded the MTD level in both sexes and produced a marked depression of body weight gain, food intake, food consumption ratios, and increased water consumption. In addition, changes to hematology parameters indicated reversible hypochromia, anisochromia, and microcytosos of red blood cells. Changes to plasma levels of proteins, glucose, bilirubin, cholesterol, and triglycerides were noted and increased activities of liver enzymes were indicative of hepatotoxic and hepatotropic effects. This was confirmed by major Histopathologic findings of hepatocellular hypertrophy and necrosis. At 1000 ppm, slight hepatotropic effects were noted in both sexes. Slight nephrotropic effects in males were typical for an alpha-2-microglobulin accumulation. In some males slight hypocellulrity of the bone marrow was observed. Reversibility was incomplete for hepatotropic effects, but was generally more pronounced in males than in females.
FOB, motor activity data, and histology of nervous tissues revealed no evidence of a neurotoxic potential of the test article.
It can be inferred from the observations made during the above study, that a “no-observable-effect level” for CA 2343 A when offered to rats continuously in their food over a period of 4 weeks is 200 ppm, corresponding to a mean daily intake of 16.3 and 15.7 mg/kg/d in males and females, respectively.

Executive summary:

Purpose of this study was to investigate the toxicity of CA 2343A (intermediate CGA 293343) to male and female rats when administered orally by gavage for 28 consecutive days.

Groups of five male and female Han Wistar rats were dosed orally with 0 (control), 50, 200, 1000 and 5000 ppm for 28 consecutive. Additionally, there were 5 males and 5 females each in the control and the high dose group for recovery (4 weeks period) evaluation.

Clinical observations, body weights, food consumption and FOBs were measured throughout the study. At the end of the study the animals were killed and examined macroscopically and microscopically. Cardiac blood samples were taken for clinical pathology. 

Under the conditions of the test, treatment with 5000 ppm CA 2343 A exceeded the MTD level in both sexes and produced a marked depression of body weight gain, food intake, food consumption ratios, and increased water consumption. In addition, changes to hematology parameters indicated reversible hypochromia, anisochromia, and microcytosos of red blood cells. Changes to plasma levels of proteins, glucose, bilirubin, cholesterol, and triglycerides were noted and increased activities of liver enzymes were indicative of hepatotoxic and hepatotropic effects. This was confirmed by major histopathologic findings of hepatocellular hypertrophy and necrosis. At 1000 ppm, slight hepatotropic effects were noted in both sexes. Slight nephrotropic effects in males were typical for an alpha-2-microglobulin accumulation. In some males slight hypocellulrity of the bone marrow was observed. Reversibility was incomplete for hepatotropic effects, but was generally more pronounced in males than in females.

FOB, motor activity data, and histology of nervous tissues revealed no evidence of a neurotoxic potential of the test article.

It can be inferred from the observations made during the above study, that a “no-observable-effect level” for CA 2343 A when administered to rats continuously in their food over a period of 4 weeks is 200 ppm, corresponding to a mean daily intake of approx. 16 mg/kg body weight.