Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
GLP compliance:
no
Test type:
traditional method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF/8801
- Purity test date:28th calendar week 1988

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stability was ensured for at least the study period.

OTHER SPECIFICS: Purity >95%

Test animals

Species:
rat
Strain:
other: SPF Wistar/Chbb: THOM
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, 0-7950 Biberach,FRG
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: Males - 267 ± 10.9 g; Females - 185 ± 7.4g;
- Housing: groups of five in cages type DK III of Becker
- Diet: KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmuhle AG, CH-4303 Kaiseraugst, Switzerland
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 hrs

Administration / exposure

Route of administration:
other: dust aerosol
Type of inhalation exposure:
nose/head only
Vehicle:
other: 1 wt% of Aerosil (Groups 1-4, 6). Group 5 - unchanged
Mass median aerodynamic diameter (MMAD):
> 1.3 - < 7.4 µm
Geometric standard deviation (GSD):
>= 2.2 - <= 4.3
Remark on MMAD/GSD:
Group 1: 1.6μm, GSD:3.5;
Group 2: 1.4μm, GSD:2.6;
Group 3: 1.5μm, GSD:3.6;
Group 4: 1.3μm, GSD:4.3;
Group 5: 7.4μm, GSD:2.2;
Group 6: 3.3μm, GSD:2.8;

Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head-nose inhalation system INA 20 (glass-steel construction, BASF Aktiengesellschaft

- Exposure chamber volume: 55L

- Method of holding animals in test chamber: The animais were restrained in tubes and their snouts projected into the inhalation chamber.

- Source and rate of air: Test groups 1 - 4 and 6: 2,000 L/hr; Test group 5: 1,500 L/hr

- System of generating particulates/aerosols: A dust aerosol was generated by means of a (i) solid particle generator (brush-generator. Technical University of Karlsruhe/BASF) (test group 5) (b) dosing-wheel dust generator (test groups 1-4 and 6)

- Method of particle size determination:
Equipment
- Stack Sampler Mark III (Andersen)
- Vacuum Compressed Air Pump - Sampling probe (internal diameter 6.9 mm)
- Balance: Mettler AE 240 and Sartorius M3P
- Evaluation unit (IBM-PC with software PGA)

Procedure
Test group 1:
The sampling was started on the same time as the exposure. The dust aerosol generation was stopped after the end of the particle size sampling (after 8 hours).

Test group 2:
The sampling was started on the same time as the exposure and was stopped after exposure.

Test groups 3 - 6:
30 minutes after the beginning of the test at the earliest, one sample was taken per test group for the particle size analysis.

Test groups 1 - 6:
Before the sampling. the impactor was equipped with glass-fiber collecting discs and a backup particle filter. The impactor was connected to the pump and the test apparatus, and one sample (180 - 1,440 L) was taken. The impactor was taken apart, and the collecting discs and the backup particle filter were weighed. The contents of the pre-impactor as well as the amounts of the material adsorbed on the walls of the impactor and in the sampling probe (wall losses) were also determined quantitatively.

- Treatment of exhaust air: By means of an exhaust air system the pressure ratios in the inhalation system were adjusted in such a way that the amount of exhaust air was abaut 5 - 10% lower (excess pressure). This ensured that the mixture of test substance and air was not diluted with laboratory air in the breathing zones of the animais.

- Temperature, humidity, pressure in air chamber: The exposure System was placed in an air-conditioned laboratory. Temperatures in the exposure system were 19- 25°C.

TEST ATMOSPHERE
- Brief description of analytical method used:
Apparatus:

- Vacuum compressed air pump (Millipore)
- Filtration equipment with probe, internal diameter: 7 mm, (Millipore)
- Filter: MN 85/90 Bf (d =4.7 cm)
- Sampling velocity: 1.25 m/s
- Sampling amount: 18 L - 150 L
- Sampling position: immediately adjacent to the animals' noses
- Sampling frequency: 1 sample per concentration group about hourly

Gravimetric determination of the inhalation atmosphere concentration:

Equipment: balance: Mettler AE 240

The preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a volume of the dust aerosol was drawn through the filter. The dust concentration in mg/l was calculated from the difference between the preweight of the filter and the weight of the filter after sampling. with reference to the sample volume of the inhalation atmosphere. The
concentrations were corrected for the amount of the added
excipient (test groups 1 - 4 and 6).

- Samples taken from breathing zone: Yes

VEHICLE
- Composition of vehicle (if applicable): Aerosil (Synthetic Amorphous Silica; CAS # 7631-86-9, 112945-52-5, 112926-00-8). The data owner of this study (BASF) provided a document that summarises the vehicle properties (see GPS Safety Summary Silicon Dioxide-EVONIK)
- Concentration of test material in vehicle (if applicable): 1%
- Justification of choice of vehicle: Used to achieve a more uniform dust concentration in air

TEST ATMOSPHERE (if not tabulated)
The test substance used in test groups 1 - 4 and 6 was mixed with 1 wt% of Aerosil in order to achieve a more uniform dust concentration in air and a cyclonic separator was connected down-stream with the generator.
The test substance used in test group 5 was unchanged. The concentration was adjusted by varying the piston feed of the dust reservoir and by varying the brush rotation (test group 5) or the rotation of the metering disc (test groups 1 - 4 and 6).

- Particle size distribution:
Group 1: A respirable dust aerosol fraction that might reach the alveolar region of 95% was obtained.
Group 2: A respirable dust aerosol fraction that might reach the alveolar region of 99% was obtained.
Group 3: A respirable dust aerosol fraction that might reach the alveolar region of 94% was obtained.
Group 4: A respirable dust aerosol fraction that might reach the alveolar region of 93% was obtained.
Group 5: A failure in generation technique produced an insufficient particle size. Therefore, the results of this test group are not used for evaluating the toxicity of the substance.
Group 6: A respirable dust aerosol fraction that might reach the alveolar region of 88% was obtained.
Refer to the test report for detailed information on particle size distribution in Section 3.3.3.

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
Group 1: 1.6μm, GSD:3.5;
Group 2: 1.4μm, GSD:2.6;
Group 3: 1.5μm, GSD:3.6;
Group 4: 1.3μm, GSD:4.3;
Group 5: 7.4μm, GSD:2.2;
Group 6: 3.3μm, GSD:2.8;

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Concentrations:
The nominal concentration (mg/l) was calculated from the amount of substance consumed and the air flow.
Group 1:0.30
Group 2: 0.36
Group 3: 0.89
Group 4:3.02
Group 5: 1.64 (disregarded due to a failure in the generation technique that produced an insufficient particle size)
Group 6: 3.35

Analytical concentrations:
Group 1: 0.006 ± 0.0003
Group 2: 0.012 ± 0.018
Group 3: 0.043 ± 0.003
Group 4: 0.11 ± 0.008
Group 5:0.26 ± 0.013 (disregarded due to a failure in the generation technique that produced an insufficient particle size)
Group 6: 0.29 ± 0.007
No. of animals per sex per dose:
5 male and 5 female per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days (all groups except 5)
- Frequency of observations and weighing: The body weight of the animais was checked before the beginning of the test, after 7 days and at the end of the observation period. Clinical findings were recodled several times during exposure and at least once an each workday in the observation period. A check for dead animais was made daily.
- Necropsy of survivors performed: Yes

Results and discussion

Effect levelsopen allclose all
Sex:
male/female
Dose descriptor:
LC50
Effect level:
0.089 mg/L air
95% CL:
> 0.044 - < 0.256
Exp. duration:
4 h
Sex:
male
Dose descriptor:
LC50
Effect level:
0.055 mg/L air
95% CL:
> 0.015 - < 0.332
Exp. duration:
4 h
Sex:
female
Dose descriptor:
LC50
Effect level:
0.136 mg/L air
Exp. duration:
4 h
Mortality:
Group 1: 0/10 deaths
Group 2: 2/10 deaths
Group 3: 2/10 deaths
Group 4: 7/10 deaths
Group 5: 0/10 deaths (disregarded)
Group 6: 7/10 deaths

Clinical signs:
Group 1: After 1 hr, all animals had accelerated respiration. From Day 3 -14, no signs were evident in any animals.
Group 2: After 15 mins, all animals had restlessness which ceased after 30 mins; after 30 mins, all animals had accelerated respiration which decreased to 5 animals after 4 hrs. Four animals had irregular respiration after 4 hrs. At 3 hrs, 5 animals had reddish eye discharge. From Day 4 -14, no signs were evident in 8 animals. Other signs evident from day 0-1 were squatting position, ruffled fur, unsteady gait and deteriorated general state.
Group 3: In under15 mins, all animals had restlessness which ceased after 30 mins; after 30 mins, all animals had accelerated respiration which decreased to 6 animals after 4 hrs. Three animals had irregular respiration after 4 hrs. From Day 2 -14, no signs were evident in 8 animals. Other signs evident from day 0-1 were squatting position, ruffled fur, high stepping gait gait and deteriorated general state
Group 4: In under15 mins, all animals had restlessness which ceased after 30 mins; after 30 mins, all animals had accelerated respiration which decreased to 8 animals after 2 hrs. By 4 hrs, 3 animals had irregular respiration, 3 animals whooping respiration and 1 animal shallow respiration. From Day 5 -14, no signs were evident in 3 animals. Other signs evident from day 0-4 were acceleareted respiration, abdominal position, squatting position, ruffled fur, unsteady gait, uring contaaminated fur and deteriorated general state.
Group 5: disregarded
Group 6: In under15 mins, all animals had restlessness which ceased after 30 mins; after 30 mins, all animals had accelerated respiration which decreased to 3 animals by 3 hrs. By 4 hrs, 6 animals had irregular respiration. From Day 6 -14, no signs were evident in 3 animals. Other signs evident from day 0-3 were acceleareted respiration, squatting position, ruffled fur, high stepping gait, and deteriorated general state.
Body weight:
The body weight gain of male rats in test groups 1 – 3 and 5 and female rats in the test groups 1, 5, 6, compared with a historical control collective, was not affected by the substance over the total observation period.

The body weight gain of female rats in the test group 4, compared with a historical control collective, was retarded in the first week of the observation period and adjusted to normal in the second week of the observation period.

The body weight gain of female rats in the test groups 2 and 3, compared with a historical control collective, was retarded in the second week of the observation period.

The body weight gain of male animals in the test groups 4 and 6 could not be evaluated because only one animal survived.
Gross pathology:
Animais that died spontaneously/sacrificed in a moribund state:
General congestion.
Lungs: focal hyperemia, some animals with emphysema.
Adrenais: dark in same animals.

Sacrificed animals: No pathalogic findings noted.

Applicant's summary and conclusion

Interpretation of results:
Category 2 based on GHS criteria
Conclusions:
In an acute inhalation study in rats, the LC50 (male/female) of N,N′-m-phenylenedimaleimide was 0.089 mg/l (0.044 mg/l - 0.256 mg/l).
Executive summary:

In an acute inhalation toxicity study (1744), groups of young adult Wistar rats (5/sex) were exposed by inhalation route to N,N′-m-phenylenedimaleimide  (>95%) in 1% Aerosil for 4 hours (head/nose only) at concentrations of  0.006, 0.012, 0.043, 0.11, 0.26 and 0.29 mg/l.  Animals were then were observed for 14 days.

LC50 Males = 0.055 mg/l (0.015 mg/l - 0.332 mg/l)

LC50 Females =  0.136 mg/l

LC50 Combined = 0.089 mg/l (0.044 mg/l - 0.256 mg/l)

The results from the 0.26 mg/l dose were disregarded due to a failure in the generation technique that produced an insufficient particle size. The MMAD for the other tests groups was 1.3 μm – 3.3 μm and GSD was 2.6 – 4.3. A respirable dust aerosol fraction that might reach 88 -99% of the alveolar region was obtained. As the dosing increased, the number of mortalities increased (0.012, 0.043 mg/l, 2/10 deaths by Day 1; 0.11, 0.29 mg/l, 7/10 deaths by Day 2). There was also a dose-dependent increase in clinical signs including effects on respiration, squatting position, ruffled fur, high stepping gait, and deteriorated general state. The body weight gain of male rats in test groups 1 – 3 and 5 and female rats in the test groups 1, 5, 6, compared with a historical control collective, was not affected by the substance over the total observation period. The body weight gain of female rats in the test group 4, compared with a historical control collective, was retarded in the first week of the observation period and adjusted to normal in the second week of the observation period. The body weight gain of female rats in the test groups 2 and 3, compared with a historical control collective, was retarded in the second week of the observation period. The body weight gain of male animals in the test groups 4 and 6 could not be evaluated because only one animal survived. In animals that died spontaneously or were sacrificed in a moribund state, pathological findings were general congestion, focal hyperemia in lungs and some animals with emphysema and dark adrenals. In sacrificed animals there were no pathological findings noted.

This acute inhalation toxicity test in rats is acceptable and satisfies the guideline requirement for an OECD 403 study.