2-nitro-4-(trifluoromethyl)benzonitrile

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun - 12 Jul 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

adopted 07 Sep 2009

Deviations:

yes

Remarks:

no nominal concentrations were determined

GLP compliance:

yes

Remarks:

The test complied with the Principles of Good Laboratory Practices (GLP) of the Certification and Accreditation Administration of the People’s Republic of China (2013 revised edition).

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-nitro-4-(trifluoromethyl)benzonitrile

EC Number:

212-298-1

EC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Cas Number:

778-94-9

Molecular formula:

C8H3F3N2O2

IUPAC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF grade

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Liaoning Changsheng Biotechnology Co., Ltd., Liaoning, China
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 305.63 g - 343.40 g (males), 219.94 g - 252.91 g (females) at grouping
- Fasting period before study: no
- Housing: individually in stainless-steel suspended cages (L 32.0 cm x W 28.2 cm x H 20.2 cm) affixed onto racks (L 167.3 cm x W 70.4 cm x H 170.9 cm)
- Diet: SPF rat/mice maintenance feed (Beijing Keao Xieli Feed Co., Ltd., China); ad libitum
- Water: purified water; ad libitum
- Acclimation period: 5 days, including in the holding device for 1 h and 2 h on acclimation days 3 and 5, respectively.
- Microbiological status when known: SPF grade
- Method of randomisation in assigning animals to test and control groups: 24 animals (half females and half males) were allocated to four groups using a random hierarchical grouping method and body weight did not exceed ± 20% of body weight for animals of the same gender in the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.00 - 23.20 (actual measured temperature)
- Humidity (%): 41.29 - 70.00 (actual measured relative humidity)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 20 Jun to 12 Jul 2019

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

other: mouth/nose

Vehicle:

other: Dimethyl sulphoxide (DMSO)

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Geometric standard deviation (GSD):

>= 1.5 - <= 3

Remark on MMAD/GSD:

figures in ranges

Every two hours, MMAD was measured once and GSD was calculated. Average MMAD value of the first group was 2.91 μm; average MMAD value of the second group was 1.90 μm; average MMAD value of the third group was 2.89 μm and average MMAD value of the fourth group was 1.90 μm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: HOPE-MED 8050F/51E/52G mouth/nose exposure apparatus
- Exposure chamber volume: not reported
- Method of holding animals in test chamber: The animals were secured in the holding device and the holding devices were secured radially for exposure at the inhalation chamber port.
- Source and rate of air: not reported
- System of generating particulates/aerosols: Test sample solution or vehicle was passed through the peristaltic pump into the liquid aerosol generator, compressed air was mixed with the test sample solution or vehicle then collided with a metal sheet to form a liquid aerosol, and entered the inhalation chamber.
- Method of particle size determination: measured by a particle size spectrometer (HOPE-MED CLA (Promo 2000) model)
- Temperature, humidity, pressure in air chamber: 22.2 - 23.3 °C, 58.4 - 60.0%, pressure not reported

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: gravimetric filter method: The sampler was used to sample the test sample aerosol, the sampling flow rate was 1 L/minute and the sampling time was one minute. The difference in the weight of the filter and the holding device before and after sampling was used to calculate aerosol concentration. Aerosol concentration was measured once every hour.
- Samples taken from breathing zone: not reported
- Time needed for equilibrium of exposure concentration before animal exposure: not reported

VEHICLE
- Concentration of test material in vehicle: first and second group: a 50% test sample solution (W/W) was prepared from test sample and DMSO
- Justification of choice of vehicle: After the test sample was crushed into pieces using a planetary ball mill, the test sample changed colour. After the test sample was dissolved in DMSO, it yielded the liquid aerosol.
- Lot/batch no.: 20190315

TEST ATMOSPHERE
- Particle size distribution: see table 1 under "any other information on materials and methods incl. tables"

CLASS METHOD
- Rationale for the selection of the starting concentration: based on the results of a pre-test (see section "results and discussion - preliminary study").

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric filter method

Duration of exposure:

4 h

Concentrations:

1 and 5 mg/L
Actual mean concentrations: 5.269 mg/L (first group); 5.140 mg/L (second group); 1.034 mg/L (third group); 1.020 mg/L (fourth group)

No. of animals per sex per dose:

3

Control animals:

yes

Remarks:

for each dose one vehicle control group was designed (second and fourth test groups)

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: On the day of exposure (Day 0), moribundity observations were performed once after exposure. On Days 1 - 14 observations were performed twice daily. Animals were weighed individually prior to exposure on the day of exposure (Day 0), Day 1, Day 3, and Day 7, and subsequently once a week, and body weights were recorded. Animals that died or were euthanised after Day 1 were weighed.
- Necropsy of survivors performed: Yes: Gross necropsies were performed on all exposed animals, including animals that died in the test period.
- Clinical signs including body weight: Clinical signs were observed prior to exposure on the day of exposure (Day 0), at 0 h, 1 h, and 2 h after exposure ended. On Day 1 to Day 14 symptoms were observed once daily. Changes to animal fur, the eyes, mucous membranes, respiratory system, circulatory system, and nervous system were observed and recorded in detail, particularly changes in limb movements and behaviour. Each animal’s toxicity symptoms and the times of their occurrence and disappearance were meticulously recorded. Attention was paid to the presence or absence of such symptoms as tremors, convulsions, salivation, diarrhoea, sleepiness, and coma.

Statistics:

not performed

Results and discussion

Preliminary study:

In a pre-test, six SD rats were exposed to the test sample aerosol at 1.5 mg/L for 4 h and no animals died. In accordance with the Chinese 'Chemical Test Methods - Health Effects Volume (second edition)', test sample aerosol concentration of 5 mg/L (± 20%) was selected as the starting concentration. If 3 - 6 animals died at 4 h of exposure to the 5 mg/L test sample aerosol, then 4 h of exposure to the 1 mg/L (± 20%) test sample aerosol concentration was used for the test.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

ca. 1.034 - ca. 5.269 mg/L air

Based on:

test mat. (dissolved fraction)

Exp. duration:

4 h

Mortality:

5 mg/L: 2/3 males and 3/3 females died
1 mg/L: 0/3 males and 0/3 females died

No mortality occurred in the vehicle control groups.

Clinical signs:

other: please refer to "other findings".

Body weight:

First group (5 mg/L test item): 3 animals (2 males and 1 female) died prior to weighing on the first day, 3 animals (1 male and 2 females) lost weight after exposure and the one surviving male gained weight before the test ended.
Second group (5 mg/L vehicle): 4 animals (3 males and 1 female) lost weight after exposure and gained weight before the test ended, and 2 males gained weight continuously.
Third group (1 mg/L test item): All animals lost weight after exposure and gained weight before the test ended.
Fourth group (1 mg/L vehicle): 3 animals (2 males and 1 female) lost weight after exposure and gained weight before the test ended and 3 animals (1 male and 2 females) gained weight continuously.

For details on body weights, please refer to Table 3 under section "Any other information on results incl. tables".

Gross pathology:

First group (5 mg/L test item): 5 animals (2 males and 3 females) presented greyish-white colour changes in the lungs
In the remaining treatment groups, no abnormalities were seen in any of the animals.

Other findings:

Clinical observations (also refer to Table 2)
5 mg/L: 2 males and 3 females had respiratory anomalies and laid on their sides, and one male exhibited spontaneous reduction in spontaneous movement
5 mg/L: 2/5 males and 3/5 females had respiratory anomalies and laid on their sides
In the other dose group and in the control group, no clinical effects were observed.

Any other information on results incl. tables

Table 2: Table for acute inhalation toxicity

Target Concentration [mg/L air]	Mortality	Clinical Signs
	N*	N*
Males
5 (test sample)	2/3	2/3
5 (vehicle)	0/3	0/3
1 (test sample)	0/3	0/3
1 (vehicle)	0/3	0/3
Females
5 (test sample)	3/3	3/3
5 (vehicle)	0/3	0/3
1 (test sample)	0/3	0/3
1 (vehicle)	0/3	0/3

*Number of animals/ number of animals used

 

Table 3: Average Body Weight Results

Average concentration (mg/L)	Sex	Average body weight (g) ± standard deviation
		Grouping	0 d	1 d	3 d	7 d	14 d
5.269 (test sample)	m	323.72 ± 13.97	323.72 ± 13.97	291.77	255.57	291.24	388.83
	f	236.68 ± 9.79	236.68 ± 9.79	222.90 ± 8.32	-	-	-
5.140 (vehicle)	m	324.64 ± 15.32	333.93 ± 11.93	324.41 ± 15.24	332.76 ± 24.47	357.32 ± 21.52	394.05 ± 16.70
	f	236.49 ± 9.40	239.14 ± 7.94	237.67 ± 9.74	246.92 ± 13.44	256.94 ± 10.95	267.93 ± 15.44
1.034 (test sample)	m	323.93 ± 13.58	342.04 ± 15.21	313.81 ± 0.99	329.17 ± 5.01	363.81 ± 7.60	397.82 ± 20.06
	f	236.47 ± 11.30	242.00 ± 12.71	226.79 ± 11.00	239.23 ± 7.96	252.02 ± 14.89	271.50 ± 11.81
1.020 (vehicle)	m	324.16 ±  18.89	340.70 ± 23.53	332.22 ± 21.81	343.24 ± 22.13	359.85 ± 17.25	386.36 ± 33.09
	f	235.61 ± 16.54	240.58 ± 16.59	241.56 ± 19.58	246.63 ± 24.21	262.55 ± 35.90	280.34 ± 29.39

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Acute Tox 4 (inhalation, H332) according to Regulation (EC) No 1272/2008

Conclusions:

In this acute inhalation toxicity study on 2-nitro-4-(trifluoromethyl)benzonitrile, the half-lethal concentration (LC50) in SD rats after 4 h mouth/nose exposure was within the range of 1.034 ± 0.041 to 5.269 ± 0.271 mg/L. According to Regulation (EC) No 1272/2008, the substance fulfills the criteria for Acute Tox 4 (inhalation, H332).