Benzenamine, 4-fluoro-N-(1-methylethyl)-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Sep - 22 Sep 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Adopted in 1981

Deviations:

yes

Remarks:

Humidity values were below guideline values due to use of conditioned dry air for dispersion of test substance. Particle size distribution was measured once (not twice) during the 4 h exposure. These minor deviations have no impact on the study outcome.

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-100-7

EC Name:

-

Cas Number:

70441-63-3

Molecular formula:

C9H12FN

IUPAC Name:

4-fluoro-N-(propan-2-yl)aniline

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF bred Wistar rats (strain: Hsd Cpb:WU (SPF)) were obtained from Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2 - 3 months
- Mean body weight at study initiation: males: 203 g; females: 182 g
- Housing: Animals were housed singly in conventional Makrolon® Type II cages. Bedding consisted of type S 8/15 low-dust wood granulate from Ssniff, Soest/Westfalen, Germany.
- Historical data: Animals of this strain have been used at Bayer AG in toxicological studies for years. Historical data on their physiology, diseases and spontaneous alterations are available.
- Diet: standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Lage, Germany), ad libitum. The nutritive composition and contaminant content of the standard diet was checked regularly by random sampling by the Laboratory Animal Services, Bayer AG.
- Water: drinking quality tap water, ad libitum
- Acclimation period: Animals were acclimatized to the animal room conditions for at least 5 days before use.
- Microbiological status: SPF bred rats
- Method of randomisation in assigning animals to test and control groups: A computerized list of random numbers served the purpose to assign animals at random to the treatment groups.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 Sep 1998 To: 22 Sep 1998

Administration / exposure

Route of administration:

inhalation: mixture of vapour and aerosol / mist

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 3.57 - <= 4.13 µm

Geometric standard deviation (GSD):

>= 1.9 - <= 2.13

Remark on MMAD/GSD:

1925 mg/m³: aerosol with MMAD = 4.13 µm and GSD = 2.13
3076 mg/m³: aerosol with MMAD = 3.57 µm and GSD = 1.90

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: aluminum inhalation chamber
- Exposure chamber volume: 3.8 L
- Method of holding animals in test chamber: plexiglas exposure tubes (nose-only exposure)
- Source and rate of air (airflow): dry conditioned air with an inlet air flow of 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- System of generating vapor atmosphere: Under dynamic conditions the vapor phase of the test substance was conveyed into the intake of the cylindrical inhalation chamber. Dry conditioned air was fed through the liquid of the test substance contained in two glass bubblers (content: 100 g/bubbler). Without further dilution the saturated vapor atmosphere was conveyed into the inhalation chamber.
- System of generating particulates/aerosols: Under dynamic conditions the test substance was nebulized into a baffle (pre-separator) from which the substance was conveyed into the intake of the cylindrical inhalation chamber. The test substance was nebulized using a binary nozzle and conditioned compressed air (15 L/min; dispersion pressure 600 kPa). The test substance was fed into the nozzle by an infusion pump equipped with 50 mL glass syringe (Braun Melsungen).
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS critical orifice, low-pressure critical orifice cascade impactor (Hauke, Gmunden, Austria). The individual impactor stages had been covered by an aluminum foil and glass fiber filter which were subjected to gravimetric analysis. An adhesive stage coating (silicone spray) was therefore not used to prevent particle bounce and re-entrainment. Gravimetric analyses were made using a digital balance.
- Treatment of exhaust air: The exhaust air was purified via cotton-wool/HEPA/char coal filters. These filters were disposed of by Bayer AG.
- Temperature, humidity in air chamber: 21.1 - 23.9 °C, 3.3 - 13.5 %

TEST ATMOSPHERE
- Brief description of analytical method and equipment used:
Vapor (low dose group): The test substance was sampled using gas bubblers filled with ethanol and were quantified analytically by gas chromatography (GC) using an flame ionization detector (FID). Between the glass bubblers and gas metering device a cool-trap was used to scrub volatile organic constituents from the sampling train to prevent sampling errors.
Aerosol (mid and high dose group): The test atmosphere consisting an equilibrium of vapor and aerosol phases was determined by gas chromatography (GC). Samples were taken by using Florisil filled tubes and/or additional gas bubblers (to check for quantitative absorption in one adsorbent or the other). For reference/calibration purposes the test substance was used.
- Samples taken from breathing zone: yes

VEHICLE
The test substance was evaporated or aerosolized neat without any vehicle. For aerosol generation, the test substance was nebulized using a binary nozzle and conditioned compressed air.

TEST ATMOSPHERE
- Particle size distribution: Mid concentration group (1.925 mg/L): 3.1 % particel mass <1.0 µm; 33.7 % particle mass <3.0 µm; 60.1 % particle mass <5.0 µm. High concentration group (3.076 mg/L): 2.5 % particel mass <1.0 µm; 39.3 % particle mass <3.0 µm; 69.9 % particle mass <5.0 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Mid concentration group (1.925 mg/L): MMAD = 4.13 µm and GSD = 2.13. High concentration group (3.076 mg/L): MMAD = 3.57 µm and GSD = 1.90

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

nominal: 0.800, 1.500, 5.000 mg/L
analytical: 0.510, 1.925, 3.076 mg/L

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The appearance and behavior of each rat were examined several times on the day of exposure and at least once daily thereafter. Body weights were measured before exposure, on days 3 and 7, and weekly thereafter. Individual weights were also recorded at death, if applicable.
- Necropsy of survivors performed: yes
- Clinical signs including body weight: Cage-side observations included, but were not limited to, changes in the skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration. The following reflexes were tested: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).
- Other examinations performed: The rectal temperatures were measured directly after cessation of exposure.

Statistics:

Necropsy: Respiratory tract findings of survivors were evaluated statistically using pair-wise Fisher test after the RxC chi-squared test. The Fisher test was only performed if differences occurred between groups in the RxC chi-squared test or if the frequency value was <5 (Gad and Weil, 1982). For calculation of the unilateral p value a symmetrical distribution was assumed.
Body weight (bw): Means and single standard deviations of bw were calculated. Since in acute studies individual group means may differ prior to the first exposure, the bw gain was statistically evaluated for each group using one-way ANOVA.
Physiological data: Data of rectal temperature measurements were evaluated using the ANOVA procedure.
Calculation of LC50: The LC50 calculation was performed by computer according to Rosiello et al. (1977) and Pauluhn (1983). If only two pairs of values with >0 % lethality and <100 % are available then the first linear approximation is based on these values and a χ²-homogeneity test is not performed. In this case the interpolated concentration at 50 % lethality is designated the approximate LC50. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
Analysis of variance (ANOVA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-α) = 95 % (p = 0.05). The test for the between-group homogeneity of the variance employed Box's test if more than two study groups were compared with each other. If the F-test shows that intra-group variability > inter-group variability, this is indicated as "no statistical difference between the groups". If a difference was found then a pairwise post-hoc comparison was conducted (one- and two-sided) (Games and Howell modification of Tukey-Kramer test).

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

2.58 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Control group: All animals survived until scheduled sacrifice.
Low concentration group (0.510 mg/L air): No mortality was noticed.
Mid concentration group (1.925 mg/L air): No mortality was noticed.
High concentration group (3.076 mg/L air): All males died within 1 - 2 days, whereas 3/5 females died wiithin 2 - 4 days.

Clinical signs:

other: Starting from the lowest tested concentration (0.510 mg/L): piloerection, reduced motility, and bradypnea

Remarks:

At higher concentrations, additionally: ungroomed hair-coat, high-legged gait, limp, labored breathing pattern, cyanosis, palpebral closure, nostrils with red encrustations, emaciation, prostration, and apathy.

Body weight:

As compared to the control group, the animals of the groups exposed to the test substance showed a transient decrease of body weight and body weight gain regarding in particular the females of the mid and the males and females of the high concentration groups.

Gross pathology:

- Animals sacrificed at the end of the observation period:
In rats exposed to the test substance a conclusive, concentration-dependent increased incidence of macroscopic findings could not be ascertained. Therefore, the discolorations of lungs observed are not considered to be causally related to the exposure to the test substance.
Findings such as mild discolorations of lungs and other parenchymatous organs are often observed in control animals euthanized with pentobarbital.
- Animals that died intercurrently: Lungs slightly collapsed, dark-red discolorations; white foamy content in trachea; black content (mucus) in gastrointestinal tract.

Other findings:

Reflex measurements
A battery of reflex measurements was made on the first post-exposure day. Some rats of the mid and high concentration group experienced decreased reflexes (grip strength, tonus, startle reflex, light reflex, tail pinch and righting response). The animals of the remaining groups were inconspicuous.

Rectal temperature
Statistical comparisons between control animals with those in the exposure groups revealed statistically significant differences, i.e., the rats exposed to the test substance experienced a decrease in body temperature (down to 27.6 °C in high concentration animals, versus approximately 38 °C for the control group).
According to the study report, the difference in body temperature may reflect a more labile thermoregulatory physiology among rodents, as a reaction toward inhalative exposure to respiratory irritating substances.

Any other information on results incl. tables

Table 1. Results of acute inhalation toxicity testing with Wistar rats exposed to N-Isopropyl-4-fluoroaniline

Test substance concentration [mg/L air]	Toxicological results*	Onset and duration of clinical signs	Time of death	Mortality (%)	Mean rectal temperature (°C)
Males
0	0/0/5	---	---	0	38.1
0.51	0/5/5	Day 0	---	0	34.9**
1.925	0/5/5	Day 0 - 7	---	0	32.4**
3.076	5/5/5	Day 0 - 2	Day 1 - 2	100	27.6**
Females
0	0/0/5	---	---	0	38.3
0.51	0/4/5	Day 0	---	0	35.1**
1.925	0/5/5	Day 0 - 4	---	0	33.0**
3.076	3/5/5	Day 0 - 5	Day 2 - 4	60	28.7**

* first number = number of dead animals; second number = number of animals with clinical signs; third number = number of animals exposed

** = statistically significantly different from control at p <0.05

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Examining the test substance for acute inhalation toxicity in male and female Wistar rats resulted in a LC50 value of 2.58 mg/L air for the test substance as aerosol.