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

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-04-10 to 2002-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

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:

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 (lntermediate of CGA 293343); CTL test substance reference number: Y11849/001
- Substance type: organic
- Physical state: Colourless to light beige solid
- Analytical purity: 99.8 %
- Lot/batch No.: 641-11-03-0071 Milled
- Storage condition of test material: 4°C
- Source: EZA Musterversand

Test animals

Species:

rat

Strain:

other: Alpk:APfSD (Wistar derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park
- Age at study initiation: Approximately 5-6 weeks old on arrival
- Weight at study initiation: 125-175g males; 120-150g females
- Fasting period before study: no
- Housing: up to 5 per cage, sexes separately, in multiple rat racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70% (the actual humidity measured was in the range of 8-62%)
- Air changes (per hr): At least 12 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Temperature and relative humidity were measured daily outside exposure periods using maximum/minimum thermometers and dial hygrometers.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: The aerodynamic particle size distribution of each test atmosphere was measured once during each of the exposure periods on days 1, 2 and 3, and also on 13 other separate occasions during the study, using a Marple Cascade Impactor (supplied by Schaefer Instruments Limited, Wantage, Oxon, UK) which aerodynamically separates airborne particles into pre­ determined size ranges. The amount of aerosol, by weight, in each size range, was then used to calculate the aerodynamic particle size distribution of the aerosol. Using a spreadsheet, linear-regression analysis was conducted on the data. From the regression line, the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Polycarbonate tubes supplied (Battelle, Geneva, Switzerland) inserted into a PERSPEX exposure chamber. The chamber was covered with an aluminium cone and stood on an aluminium base. The atmosphere was shown to be acceptably stable over approximately 30 minutes before exposure of the test animals. During this period the holes of the exposure chamber were plugged.

- Source and rate of air: Each test atmosphere was generated using a modified Wright's dust feed. Clean, dry air (dried and filtered using equipment supplied by Atlas-Copco, Sweden) was passed through the dust feed at nominal flow rates given below (at normal temperature and pressure)

- System of generating particulates/aerosols: Each test atmosphere was generated using a modified Wright's dust feed

- Temperature, humidity, pressure in air chamber: The temperature and relative humidity in each chamber were recorded approximately every 30 minutes during exposure using a portable, digital temperature and relative humidity monitor.

- Air flow rate: Air flows were monitored and recorded at approximately 30 minute intervals using variable area flowmeters (KDG Flowmeters, Burgess Hill, Sussex, UK) and were altered as necessary to maintain the target concentrations.

Nominal generation air flow rates are shown below:
Group 1 and 2 (control): 30 litres/minute
Group 3: 3 litres/minute
Group 4: 4 or 5 litres/minute
Group 5 and 6: 30 litres/minute
The atmosphere for Groups 3 and 4 was generated using 30 l/min airflow into the stock chamber. The resultant atmosphere was passed at 3 or 4/5 l/min into the exposure chambers together with typically 18 or 8 l/min diluting air respectively to achieve and maintain the target concentrations.

- Air change rate: 12 changes per hour
- Method of particle size determination: High Performance Liquid Chromatography (HPLC)
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically: drawing each test atmosphere, at a known flow rate, for a known time, through a 25mm diameter, polyvinyl chloride (PVC) GLA 5000 filter housed in a Delrin open-faced filterholder (both filters and holders supplied by Gelman Sciences limited, Northampton, UK). The filter was weighed before and after the sample was taken.

- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The particulate concentration of each test atmosphere, close to the animals' breathing zone, was measured gravimetrically at least three times during each 6-hour exposure period.
This was done by drawing each test atmosphere, at a known flow rate, for a known time, through a 25mm diameter, polyvinyl chloride (PVC) GLA 5000 filter housed in a Delrin open-faced filterholder (both filters and holders supplied by Gelman Seiences Lirnited, Northampton, UK). The filter was weighed before and after the sample was taken. The concentration was calculated as follows:

Concentration (mg/l) = (post wt [mg] - pre wt [mg])/time [minutes] x airflow [l/min])

pre wt = weight of filter prior to sampling
post wt = weight of filter after sampling

Duration of treatment / exposure:

The animals were exposed for 6 hours per day,
5 days per week, over a period of 90 days.

Frequency of treatment:

The animals were exposed for 6 hours per day,
5 days per week, over a period of 90 days.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/l
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.01 mg/l
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.05 mg/l
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
0.25 mg/l
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale: dose range finding study; groups of 5 male and 5 female rats were exposed to atmospheres of 0, 0.05, 0.15 or 0.5 mg/l for 5 days per week over a period of 28 days for 6 hours per day. Body weights and food consumption were reduced for males exposed to 0.15 and 0.5 mg/l, and for females at 0.5 mg/l only. Histopathological effects in the nasal passages were observed in both sexes at all exposure concentrations.

The highest concentration of 0.25 mg/l chosen for the 90-day inhalation toxicity study was expected to result in toxic effects (bodyweight reduction), but not death. The intermediate concentration of 0.05 mg/l was spaced to produce a graduation of toxic effects, and the lowest concentration of 0.01 mg/l was expected to produce no evidence of toxicity.

- Rationale for animal assignment (if not random): random

- Post-exposure recovery period in satellite groups: 8 weeks

Positive control:

no positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the study and at least twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to exposure on day 1, then weekly and prior to termination

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was recorded continuously throughout the study for each cage of rats and calculated, at weekly intervals

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all study animals were examined prior to the start of exposures. The eyes of the control and highest concentration rats (groups 1 and 5) were examined during week 13. Intermediate groups (3 and 4) and recovery animals (groups 2 and 6) (week 13 and 17 respectively) were not examined since there were no findings in animals exposed to the highest concentration (group 5) at 13 weeks.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At scheduled termination of the study, all rats had blood samples collected by cardiac puncture.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: A blood film was prepared for all animals and examined where the automated analysis result suggested that this was necessary.
- Parameters examined are listed in "any other information on materials and methods incl. tables".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At scheduled termination of the study, all rats had blood samples collected by cardiac puncture.
- Animals fasted: No data
- How many animals: all animals
- Parameters examined are listed in "any other information on materials and methods incl. tables".

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All rats were killed by overexposure to halothane Ph. Bur. vapour followed by exsanguination at the end of the study. All animals were examined post mortem.This involved an external observation and detailed examination of thoracic and abdominal viscera.
Organs and tissues were preserved during the gross necroscopy according the guideline OECD 413.

Other examinations:

none

Statistics:

All data were evaluated using analysis of variance and/or covariance for each specified parameter using the MIXED procedure in SAS (1999).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance around the snout and salivation was recorded for animals exposed to 0.25 mg/l.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test substance around the snout and salivation was recorded for animals exposed to 0.25 mg/l.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly lower for animals exposed to 0.25 mg/l than controls throughout the exposure period, (maximum effect 5% in both sexes), although only occasionally statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slightly lower than controls for animals exposed to 0.25 mg/l during the majority of the exposure period, but recovered to control levels during the recovery period

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma cholesterol was lower than control in females exposed to 0.05 and 0.25 mg/l

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

After the 90 days exposure period, treatment-related lesions were confined to the nasal cavity.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths. Abnormalities generally associated with restraint (wet fur, stains around the snout and chromodacryorrhoea) were recorded for some or all animals during some or all of the exposure periods. There were no other clinical observations for animals exposed to 0.01 or
0.05 mg/l. Test substance around the snout was often recorded for animals exposed to 0.25 mg/l. In addition, salivation was recorded for animals exposed to 0.25 mg/l towards the end of each exposure period.
There were no treatment-related clinical observations recorded during the weekly clinical examinations throughout the study.

BODY WEIGHT AND WEIGHT GAIN
Bodyweights for both males and females exposed to 0.25 mg/l were slightly lower than controls throughout the exposure period, (maximum effect 5% in both sexes), although only occasionally statistically significant.
There were no effects on bodyweight for animals exposed to 0.05 or 0.01mg/l.
During the recovery period, bodyweights for females exposed to 0.25 mg/l recovered to be comparable to control. In males, bodyweights improved and were 3% lower than control at the end ofthe recovery period.

FOOD CONSUMPTION
Food consumption was slightly lower than controls for animals exposed to 0.25 mg/l during the majority of the exposure period, but recovered to control levels during the recovery period.
There were no treatment-related effects on food consumption for animals exposed to 0.01 and 0.05 mg/l compared with controls.

OPHTHALMOSCOPIC EXAMINATION
There were no abnormal eyes recorded for highest exposure concentration animals at week 13.

HAEMATOLOGY
After the 90 days exposure period, there were no treatment-related effects on the haematological or clotting parameters at any exposure concentration.
The minimally lower mean cell volume and higher prothrombin time, seen in females exposed to 0.25 mg/l after the 8 week recovery period, were notseen immediately after the exposure period and were considered to be incidental to treatment.

CLINICAL CHEMISTRY
Plasma cholesterol was lower than control in females exposed to 0.05 and 0.25 mg/l. There was evidence of a recovery towards control levels during the 8 week recovery period in females exposed to 0.25 mg/l.
After exposure to 0.25 mg/l, plasma urea and triglycerides were lower than control in males and plasma albumin and total protein were lower than controls in females. The decreases were minimal, of limited or no biological or toxicological significance and were not seen after the 8 week recovery period. After the recovery period, plasma calcium was minimally lower than control in females exposed to 0.25 mg/l. This difference was so small that it is considered to be of no toxicological significance.
Differences from control values for plasma creatinine, total bilirubin and alkaline phosphatase in males; alanine aminotransferase, aspartate aminotransferase and chloride in females at 0.01 or 0.05 mg/l were not dose-related and were considered nottobe treatment-related.

ORGAN WEIGHTS
There were no treatment-related effects on organ weights.
The slightly higher kidney weight and liver weights adjusted for bodyweight in males exposed to 0.05 g/l were not dose-related and considered to be incidental to treatment. The slightly lower liver weight seen in females exposed to 0.25 mg/l was due to the lower final bodyweight and no difference from control was seen when liver weight was adjusted for bodyweight.

GROSS PATHOLOGY
There were no treatment-related macroscopic findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
After the 90 days exposure period, treatment-related lesions were confined to the nasal cavity. Changes comprised disorganisation of the olfactory epithelium, which was characterised by a dissordered appearance, particularly in the basal areas, with or without increased prominence of the Bowman's gland ducts and occasional cysts and rosettes. Changes were most evident in the septal portions of the olfactory epithelium. This disorganisation was increased in incidence at 0.25 mg/l in both males and females and was either of a minimal or slight severity.
There were no abnormalities detected in the nasal cavity of other exposed groups and there were no treatment-related changes in any other organ examined.
After the 8 week recovery period, there was evidence of recovery of the changes in the nasal cavity in most male and female animals in the recovery group. However, in all but 2 female rats minor changes were present. In most rats these took the form of isolated cysts in the olfactory epithelium with or without occasional rosette formations. One male rat had evidence of a disorganised epithelium and 2 male rats and one female showed the presence of focal areas of hyperplasia of the olfactory epithelium.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

0.05 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly lower plasma cholesterol in females; not considered to be relevant

2

Dose descriptor:

LOAEC

Effect level:

0.25 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lower bodyweight and food consumption in both sexes exposed to 0.25 mg/l and lower plasma cholesterol in females; histopathological changes seen in the olfactory epithelium of both sexes exposed to 0.25 mg/l

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Total particulate concentration (mg/l) Mean±SD; Target concentration in brackets ( ):

Group 3: 0.011 ± 0.001 (0.01)

Group 4: 0.051 ± 0.003 (0.05)

Group 5&6: 0.255 ± 0.007 (0.25)

Aerodynamic particle size distribution

Range Median size (MMAD) µm:

Group 3: 2.05-3.16

Group 4: 2.37-3.49

Group 5&6: 2.96-4.10

Analysed atmospheric concentration (mg/1) Mean±SD; target concentration in brackets:

Group 3:0.011 ± 0.002 (0.01)

Group 4: 0.051 ± 0.004 (0.05)

Group 5&6: 0.252 ± 0.013 (0.25)

Applicant's summary and conclusion

Conclusions:

Nose-only exposure for 6 hours per day, 5 days per week over a period of 90 days to CA 2343 A (Intermediate of CGA 293343) at concentrations of 0.01, 0.05 or 0.25 mg/l resulted in lower bodyweight and food consumption and histopathological changes in the olfactory epithelium in both sexes exposed to 0.25 mg/l. There were no adverse effects of treatment in animals exposed to 0.05 mg/l (NOAEC).

Executive summary:

In a subchronic inhalation toxicity study CA 2343 A (Intermediate of CGA 293343) was administered to groups of 10 female and 10 male rats ( strain Alpk:APfSD) by dynamic nose only exposure at concentrations of 0, 0.01, 0.05 and 0.25 mg/L for 6 hours per day, 5 days/week for a total of 90 days. An additional control group and treatment group exposed to the highest concentration were included for assessment of recovery. Recovery animals were retained without treatment at the end of the 90 day exposure period for a further 8 weeks.

Analysis of the data showed that the achieved concentrations and physical characteristics of the atmospheres were satisfactory throughout the study. Both the particulate and analyzed concentrations showed that the target concentrations were achieved.

There were no deaths. Clinical observations due to restraint were seen in all animals during exposure periods. Animals exposed to 0.25 mg/l also had test substance around the snout and were salivating towards the end of most exposure periods. Salivation during and after the exposure periods is considered to reflect the irritant nature of the test aerosol.

Exposure to 0.25 mg/l CA 2343 A (Intermediate of CGA 293343) resulted in lower bodyweight and food consumption in both sexes and lower plasma cholesterol in females.

All these effects showed evidence of recovery either back to or towards control levels during the 8 week recovery period. However, there were histopathological changes seen in the olfactory epithelium of both sexes exposed to 0.25 mg/l after the 90 day exposure period, which had not fully recovered within the 8 week recovery period.

The only effect of treatment seen in animals exposed to 0.05 mg/l was slightly lower plasma cholesterol in females. This is considered to be the no observed adverse effect concentration (NOAEC).

This subchronic inhalation toxicity study in the rat is acceptable, and satisfies the guideline requirement for a subchronic inhalation study according OECD 413 in the rat.