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:

1996-07-18 till 1997-01-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

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:

yes

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:

Hill formula: C4H8N4O3 CAS 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 2343 A (Intermediate of CGA 293343)
- Substance type: mono-constituent organic substance
- Physical state: solid, fine, white powder
- Analytical purity: 96.7 %
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: P.503005
- Expiration date of the lot/batch: Date of reanalysis: December 1996
- Stability under test conditions: not reported
- Storage condition of test material: 0 - 5 °C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production CIBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: 161.1 - 214.5 g in males, 139.1 - 168.1 g in females
- Fasting period before study: diet provided ad libitum, except for overnight fasting prior to blood collection
- Housing: The experiment was carried out under specified pathogen free (SPF) standard laboratory conditions. The animals were housed individually in Macrolon cages type 3 (area: 900 square centimeters) with wire mesh tops and standardized granulated soft wood bedding. Cages
were allocated to racks by sex and group.
- Diet (e.g. ad libitum): Pelleted, certified standard diet (Nafag No. 8900 FOR GLP), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days


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


IN-LIFE DATES: From: 1996-07-25 To: 1996-10-04

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
After weighing of test item CA 2343 A, the pulverized diet was homogeneously mixed with the appropriate concentrations of the test article and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air dried and then stored in a deep freezer.


DIET PREPARATION
- Rate of preparation of diet (frequency): The approximative amount of diet needed for the whole treatment period was prepared in one batch. Food portions for one week feeding were thawed at weekly intervals.
- Mixing appropriate amounts with (Type of food): Pelleted, certified standard diet (Nafag No. 8900 FOR GLP) containing the appropriate concentrations of the test article was provided ad libitum
- Storage temperature of food: - 20 °C


VEHICLE
- Justification for use and choice of vehicle (if other than water): no vehicle used
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses of the test article content were undertaken with the diet batch prepared for the whole treatment period. These analyses were carriedout in the analytical laboratories of the Crop Protection Division, Residue Analysis, CIBA-GEIGY Ltd, Basel / Switzerland. The results thereof (Feed
Analysis Report 962044) are given in the resultss and appendix of the original study report.

Duration of treatment / exposure:

28 days

Frequency of treatment:

test item application via ad libitum diet, 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

5 males and 5 female

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
Dose level s 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 in rats (Gavage) : LD 50 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 bw and to cause no observable effects
200 ppm expected to result in a daily intake of about 20 mg/kg bw and to cause no or minimal adverse effects
1000 ppm expected to result in a daily intake of about 100 mg/kg bw and to cause minimal adverse effects
5000 ppm expected to result in a daily intake of about 500 mg/kg bw and to cause observable adverse effects , but no or few fatalities to permit a meaningful evaluation of the study.

- Rationale for animal assignment (if not random): computer-generated random numbers
- Rationale for selecting satellite groups: From t h e same batch of animals a small number was retained for possible replacement during the acclimtization period of animals deemed not suitable for study.
- Post-exposure recovery period in satellite groups: 1 month recovery period; 5 males and 5 females each in the control and high dose group for recovery evaluation.
- Section schedule rationale (if not random): random

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at AM and PM
- Cage side observations in Appendix A were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed during pretest and once weekly thereafter, at about the same time each day.


BODY WEIGHT: Yes
- Time schedule for examinations: The weight of all animals was recorded individually at weekly (midweek) weighing sessions. The 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
The food consumption was recorded weekly and was calculated for periods of one week. The calculation was based on the weight of the offered diet at the beginning of a weighing period and its difference to the re-weighed amount after several days. The food consumption ratios were calculated as mean of individual ratios according to the following formula: (weekly food consumption ( g ) -1000) / midweek body weight ( g )) x (1000 / 7)
Unit: g food/kg body weight per day
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The test article intake was calculated according to the following formula (rounded to 3 meaningful digits) : (food consumption ratio x nominal concentration (ppm)) / 1000
Unit: mg test article / k g body weight per day
An overall mean value (MEAN1) was calculated based on the nominal content of the test article in the food. Additionally, this mean value was corrected for the analytically determined test article content in the food (MEAN2).


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): yes
- Time schedule for examinations: The water consumption was recorded weekly and was calculated for periods of one week. The calculation was based on the weight of the offered water at the beginning of a weighing period and its difference to the re-weighed amount after one day.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: carried out on all surviving animals of each dose group a t the end of the treatment period, and additionally at the end of the recovery period. Blood was consistently sampled in the morning.
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, during the night before blood sampling
- How many animals:all surviving animals
- Parameters checked in section 3.9.1 of the original study report were examined


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: carried out on all surviving animals of each dose group a t the end of the treatment period, and additionally at the end of the recovery period. Blood was consistently sampled in the morning.
- Animals fasted: Yes, during the night before blood sampling
- How many animals:all surviving animals
- Parameters checked in section 3.9.2 of the original study report were examined


URINALYSIS: Yes
- Time schedule for collection of urine: carried out on all surviving animals of each dose group a t the end of the treatment period, and additionally at the end of the recovery period. Urine for analysis was collected overnight.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. Food and water was withheld during the time of urine collection .
- Parameters checked in section 3.9.3 of the original study report were examined


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observational batteries (FOBs) were conducted at weeks 4 and 8 (recovery groups only), at about the same time each day and were always conducted before the assessment of motor activity.
- Dose groups that were examined: all dose levels, and recovery groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Neurological exmainations: sensorimotor functions (approach, touch, vision, audition, pain, vestibular); autonomic functions (pupillary reflex , body
temperature); sensorimotor coordination (grip strength , landing foot splay)
FOB: a mulititude of ndividual observations, see section 3.7 of original study report
Motor activity: Horizontal activity: total distance (in cm), number of movements (counts), movement time (in sec)
Vertical activity: vertical activity (counts), number of rearings (counts), movement time (in sec)
Other parameters: time in central quadrant (centertine; in s )

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Macroscopical examination: see section 3.10.1 of original study report; microscopical examination see 3.10.2)
HISTOPATHOLOGY: Yes. At the subsequent histopathological evaluation the diagnosis or diagnoses corresponding to the mcroscopically identified lesions were correlated with the changes seen at necropsy .

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 treated group was compared to the control group either by Lepage's o r by Wilcoxon's two-sample test and tested for increasing or decreasingtrends from control up to the respective dose group by Jonckheer'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 Jonckheere test is sensitive to monotone dose-related effects.
Two-sided asymptotic p-values are reported in the " statistics" tables. Flags for significant differences between groups or trends over groups ( + or - ) are given in the "means" tables according to the specified significance level. Statistical tests and flag s used are indicated in the header of each table.
At each time point FOB data and motor activity data (session totals named as 'area under the curve' (AUC)) of treated groups were compared to the control group using multiple t tests. Raw and multiplicity - adjusted p - values based on bootstrap resampling were given.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. No clinical observation was noted except for one female of the 5000 ppm dose group showing transient hunched posture between day 13 and day 38.


BODY WEIGHT AND WEIGHT GAIN
At 5000 ppm depressed body weight gain was recorded for males (- 42 %) and females (- 47 %) and resulted in mean body weights 19 % and 14 %, respectively, below the control means at week 4. At the end of the recovery the mean carcass weight of 5000 ppm dose males were still depressed by 11 %, whereas in females complete reversibility was noted.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean food intake and the mean food consumption ratios were markedly depressed in males and females given 5000 ppm. These findings were reversible.


FOOD EFFICIENCY
Food consumption ratios were affected in high dose animals during the first treatment week. A trend towards normal values was noted during the consecutive treatment weeks as w ell as during recovery.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The overall mean water consumption was increased in animals at 5000 ppm and was still increased at the end of the recovery.


OPHTHALMOSCOPIC EXAMINATION
not examined


HAEMATOLOGY
Hematology revealed statistically significant decreased hemoglobin and MCV values and decreased MCH concentration for the 5000 ppm females. In male rats these findings were also recorded but were not statistically significant. In addition, hypochromia, anisochromia, microcytosis of red blood cells were observed in animals of both sexes. These findinds were fully reversible after recovery.


CLINICAL CHEMISTRY
Clinical biochemistry revealed lower plasma protein levels in animals given 5000 ppm. In males it was caused by a reversible decrease of plasma globulin, resulting in higher albumin to globulin ratios. Females showed decreased values of plasma albumin and globulin, whereby the changes of albumin slightly persisted after recovery. The following changes noted at the end of the treatment period were fully reversible after the recovery: lower plasma glucose, higher plasma bilirubin, higher plasma cholesterol, and slightly higher plasma triglyceride levels. Additionally, higher activities of ASAT, ALAT and gamma-glutamyl transpeptidase were observed in males.


URINALYSIS
At 5000 ppm a slightly larger amount of urine with lower relative density was noted in animals of both sexes. These changes were reversible within the recovery period.



NEUROBEHAVIOUR
There were no test substance-related effects during functional observational battery and no differences in motor activity measurements.


ORGAN WEIGHTS
no effects


GROSS PATHOLOGY
The mean carcass weights were decreased in the 5000 ppm dose males (- 20 %) and females (- 17 %), when compared with the control groups. Consequently, the mean organ weights of males (except for brain, testis, thyroid gland, and epididymis) and 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). After recovery period the mean carcass weight of 5000 ppm dose males were still depressed by 11 %. The absolute mean weights for liver (- 12 %), kidney (- 10 %), and spleen (- 14 %) were still lower than control means.
Treatment-related effects were observed in the liver. There was hepatocellular hypertrophy in animals of either sex treated with 5000 ppm and in few animals at 1000 ppm. In addition there were slight necrosis of single cells in the liver of males at 5000 ppm; slight inflammatory cell infiltration, Kupffer cell hyperplasia and deposition of pigment in animals of both sexes; and cytoplasmic inclusion bodies in male rats only. These findings were fully
reversible, except for the hepatocellular hypertrophy and inflammatory changes in the liver of either sexes and Kupffer cell pigmentation in females, findings which were only partially reversible during the recovery.


HISTOPATHOLOGY: NON-NEOPLASTIC
In the kidneys, tubular hyaline changes were noted in males given 1000 and 5000 ppm. This finding in male rats is typical for an accumulation of alpha-2-micro-globulin in renal proximal tubular epithelial cells. Tubular hyaline droplet formation in male rat kidney is considered to be male rat specific and not relevant for human health.


OTHER FINDINGS

Effect levels

1

Dose descriptor:

NOEL

Effect level:

16 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

LOAEL

Effect level:

85 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: At 1000 ppm (i.e. daily intake 85 mg/kg bw/d) microscopical changes in liver and kindney were observed, whereas at 200 ppm no adverse effects were being reported. Thus the LOAEL of 1000 ppm corresponds to a substance intake of 85 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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 approximately 16 mg/kg bw/d. A LOAEL of 85 mg/kg bw/day was being observed for both sexes, based on nominal concentrations.

Executive summary:

A 28 -d oracal acute toxicty study was carried out acc. to OECD Guideline 407. The test article CA 2343 A (Intermediate of CGA 293343, batch P. 503005, purity 96.7%) was administered in pelleted diet for 4 weeks a t nominal dietary levels of 0 , 50 , 200, 1000, and 5000 ppm (= mg/kg food) to a total of 70 albino rats. In each dose group 5 animals per sex and group were 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 11). Clinical signs, body weight, food consumption, water consumption and mortality were monitored throughout the study for all animals. Neurotoxicologic investigations were performed weekly (detailed clinical signs) and at weeks 4 and 8 (functional observational battery, motor activity). Hematological, blood chemistry and urine analyses were performed at the end of the treatment period on all animals, and at the end of the recovery period on animals of experimental group 11. At sacrifices, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation.

Under the conditions of this 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 intakes, food consumption ratios, and increased water consumption. In addition, changes to hematology parameters indicated reversible hypchromia, anisochmmia, and microcytosis 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 effecs. This was confirmed by the 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 hypocellularity 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 item.

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 approximately 16 mg/kg body weight.