N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04/08/2020 -18/12/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

EC Number:

835-183-3

Cas Number:

83420-16-0

Molecular formula:

C56 H102 Cl2 N14

IUPAC Name:

N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (SUQIAN UNITECH CORP., LTD;) and lot/batch number of test material:Batch #: 190701
- Expiration date of the lot/batch: July 24, 2021
- Purity:91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions:Test substance was expected to be stable for the duration of testing.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance, as received, was a powder. During trials a sub-sample of the test substance was ground, but this did not yield a good result; therefore the test substance was aerosolized neat for the exposures.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9-12 weeks
- Weight at study initiation: males 326.4-429.1 grams and females 218.0-289.9 grams
- Housing: Animals were group housed, except on the day of exposure, at which time they were singly housed and until the animals were deemed acceptable, based on observations, to return to group housing. Enrichment (e.g., toy) was placed in each cage and litter was changed at least once per week.
- Diet: Envigo Teklad Global 16% Protein Rodent Diet® #2016 ad libitum
- Water: Filtered tap water was supplied ad libitum
- Acclimation period: 13-37 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23ºC
- Humidity (%): 51-64%,
- Air changes (per hr): 12.
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 1.88 - ca. 2.19 µm

Geometric standard deviation (GSD):

ca. 2.25 - < 2.46

Remark on MMAD/GSD:

Main test (MMAD, GSD)
Group 1: 5.04 mg/L = 2.19 µm, 2.32
Group 2: 2.04 mg/L = 2.08 µm, 2.25
Group 3: 0.55 mg/L = 1.88 µm, 2.26
Group 4: 0.056 mg/L = 2.17 µm, 2.46

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Prior to initiation of the full inhalation study, pre-test trials were conducted to establish generation procedures to achieve, to the extent possible, the desired chamber concentration and desired particle size distribution (mass median aerodynamic diameter between 1 and 4 µm).

- Exposure apparatus: ADG Developments LTD 6.7 or 28 liter (Nose-Only Inhalation Chamber)

- Exposure chamber volume: 6.7 or 28 liter

- Method of holding animals in test chamber: A nose-only inhalation chamber was used for exposure. Animals were individually housed in polycarbonate holding tubes which seal to the chamber with an “O” ring during exposure. The base unit terminates the chamber with a 0.5-inch diameter tube for discharged air.

- Source and rate of air (airflow): Filtered generator air was supplied to the spray atomization nozzle by an air compressor, and measured with a Mass Flow Controller. Additional filtered mixing air from the same air compressor, measured with a Mass Flow Controller, was introduced into the chamber to help uniformly distribute the test atmosphere by creating a vortex at the chamber inlet. Chamber airflow was monitored throughout the exposure period and recorded periodically. The exposure was conducted under slight negative pressure.
Main test rate of airflow:
Group 1: 5.04 mg/L = 60 L per minute
Group 2: 2.04 mg/L = 60 L per minute
Group 3: 0.55 mg/L = 36 L per minute
Group 4: 0.056 mg/L = 50 L per minute

- System of generating particulates/aerosols:
The test substance was aerosolized using a Jet-Mill for 5.04 mg/L and 2.04 mg/L exposures. The test substance was delivered from the hopper using a variable speed motor and a 3/8-inch, closed pitch helix into the JetMill. Compressed generator and mixing air were both supplied. The aerosolized dust was then fed directly into the chamber through the dust outlet assembly.

The test substance was aerosolized using a modified Wright Dust Generator for 0.5 mg/L and 0.05 mg/L exposures. The test substance was packed into the dust container and compressed 500 or 1000 lbs/in2 using a lab press. The container was then fitted with a cutting head. Compressed generator and mixing air were supplied to the dust generator. The aerosolized dust was then fed directly into the chamber through the dust outlet assembly.

- Method of particle size determination:
An eight-stage 1 ACFM Westech Ambient Particle Sizing Sampler was used to assess the particle size distribution of the test atmosphere. Samples were withdrawn from the breathing zone of the animals at two intervals. The filter paper collection stages were weighed before and after sampling to determine the mass collected upon each stage. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated using two-cycle logarithmic probit axes.

- Temperature, humidity, pressure in air chamber:
The temperature and relative humidity within the exposure chamber as well as the room were monitored continuously during exposure, and were measured with a temperature-humidity monitor. Temperature and relative humidity values were recorded every 15 minutes for the first hour of exposure and approximately every 15 or 30 minutes thereafter.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: Gravimetric samples of the test atmosphere were withdrawn at 6 intervals from the breathing zone of the animals. Samples were collected using 37 mm glass fiber filters (Whatman™ GF/B) in a filter holder attached by ¼ inch Tygon® tubing to a vacuum pump. Filter papers were weighed before and after collection to determine the mass collected. This value was divided by the total volume of air sampled to determine the chamber concentration. Sample airflows were measured using a Mass Flow Controller. Refer to Appendix C for specific details on equipment.

- Samples taken from breathing zone: yes

- Time needed for equilibrium of exposure concentration before animal exposure：The exposure period was extended beyond 4 hours to allow the chamber to reach equilibrium (T99)
Group 1: 5.04 mg/L = 2.15 min
Group 2: 2.04 mg/L = 2.15 min
Group 3: 0.55 mg/L = 0.86 min
Group 4: 0.056 mg/L = 2.58 min

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetric analysis as described above

Duration of exposure:

> 241 - < 243 min

Remarks on duration:

Group 1: 5.04 mg/L = 243 min Group 2: 2.04 mg/L = 243 min Group 3: 0.55 mg/L = 241 min Group 4: 0.056 mg/L = 243 min

Concentrations:

Group 1: 5.04 mg/L (actual), 6.17 mg/L (nominal)
Group 2: 2.04 mg/L (actual), 2.77 mg/L (nominal)
Group 3: 0.55 mg/L (actual), 1.24 mg/L (nominal)
Group 4: 0.056 mg/L (actual), 0.140 mg/L (nominal)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Individual body weights of the animals were recorded prior to test substance exposure (initial) and again on Days 1, 3, 7, and 14 (terminal) or after death.
All animals were observed for mortality during the exposure period. The animals were examined for signs of gross toxicity, and behavioral changes upon removal from the exposure tube and at least once daily thereafter for 14 days or until death occurred.
- Necropsy of survivors performed: Gross necropsies were performed on all decedents and euthanized animals. Tissues and organs of the thoracic and abdominal cavities were examined.
- Clinical signs: Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.

Statistics:

Statistical analysis was limited to the calculation of the mean and standard deviation. Additionally, Probit Analysis was used for data analysis of the LC50 and confidence limit calculations (BioStat).

Results and discussion

Preliminary study:

Pre-test ([Target, Trial]; MMAD)
5 mg/L, 5.50 mg/L; 2.05 µm
2 mg/L, 2.10 mg/; 2.09 µm
0.5 mg/L, 0.55 mg/L; 1.66 µm
0.05mg/L, 0.05 mg/L; 1.94 µm

Specific details of the pre-test trials are recorded in Tables 1 through 3.

Effect levels

Effect levels 1

Sex:

male

Dose descriptor:

LC50

Effect level:

0.38 mg/L air

95% CL:

> 0.06 - < 2.31

Exp. duration:

4 h

Effect levels 2

Sex:

female

Dose descriptor:

LC50

Effect level:

> 0.056 - < 0.55 mg/L air

Exp. duration:

4 h

Mortality:

Group 1: 5.04±0.13 mg/L = 5/5 males; 5/5 females (All animals were found dead at chamber removal.)
Group 2: 2.04±0.06mg/L = 5/5 males; 5/5 females (Eight animals (5/5 males, 3/5 females) were found dead at chamber removal. Two animals died (2/5 females) within one hour of exposure to the test atmosphere.)
Group 3: 0.55±0.04 mg/L = 4/5 males; 5/5 females
Group 4: 0.056±0.016 mg/L = 0/5 males; 0/5 females

Clinical signs:

other: See below

Body weight:

Group 3: 0.55±0.04 mg/L = After initial clinical signs, the surviving animal gained body weight over the 14-day observation period.
Group 4: 0.056±0.016 mg/L = All animals gained body weight during the study.

Gross pathology:

5.04 mg/L group:Gross necropsy of the decedents revealed discoloration of the lungs and distention of the stomach and/or liquid in the trachea.
2.04 mg/L group: Gross necropsy of the decedents revealed discoloration of the lungs and/or liquid in the trachea.
0.55 mg/L group：Gross necropsy of the decedents revealed discoloration of the lungs and/or liquid in the trachea. No gross abnormalities were noted for the euthanized animal when necropsied at the conclusion of the 14-day observation period.
0.056 mg/L group：No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

Any other information on results incl. tables

Clinical signs

Group 1: 5.04±0.13 mg/L = Clinical signs were not noted due to the position in the exposure tube(s).  

Group 2: 2.04±0.06mg/L =  Clinical signs were not noted due to the position in the exposure tube(s).  

Group 3: 0.55±0.04 mg/L =  Prior to death, the animals were hypoactive and exhibited irregular respiration and/or prone posture.  Following exposure, one surviving animal was hypoactive and exhibited irregular respiration.  However, the animal recovered by Day 4 and appeared active and healthy for the remainder of the study.

Group 4: 0.056±0.016 mg/L = Following exposure, all rats exhibited irregular respiration.  However, all animals recovered by Day 3 and appeared active and healthy for the remainder of the 14-day observation period.

Applicant's summary and conclusion

Interpretation of results:

Category 2 based on GHS criteria

Conclusions:

Under the conditions of this study, the acute inhalation defined LC50 of N4, N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] by calculated Probit Analysis (BioStat) is 0.38 mg/L with 95% confidence limits of 2.31 mg/L (upper) and 0.06 mg/L (lower) in male rats. The data does not permit calculation of the LC50 for females by Probit Analysis. The LC50 for females is estimated to be between 0.056 mg/L and 0.55 mg/L.

Executive summary:

In an acute inhalation toxicity study (OECD 403/GLP), 4 groups of young adult Sprague-Dawley rats (5/sex) were exposed to a test atmosphere of N4, N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5- triazine-2,4-diamine] (91%) for 4 hours (nose only) at  mean actual concentrations of 5.04±0.13 (group 1), 2.04±0.06 (group 2), 0.55±0.04 mg/L (group 3) or 0.056±0.016 mg/L (group 4). Animals were then observed for 14 days.

LC50 Males = 0.38 mg/L (95% C.I. 0.06 - 2.31)

LC50 Females = 0.056 - 0.55 mg/L (estimate)

The MMAD was 1.88-2.19 μm and GSD was 2.25-2.46.  

In group 1, all animals were found dead at chamber removal. Gross necropsy of the decedents revealed discoloration of the lungs and distention of the stomach and/or liquid in the trachea.

In group 2, 8 animals (5/5 males, 3/5 females) were found dead at chamber removal.  Two animals died (2/5 females) within one hour of exposure to the test atmosphere. Gross necropsy of the decedents revealed discoloration of the lungs and/or liquid in the trachea.

In group 3, 4 males and 5 females died within two days of exposure to the test atmosphere.  Prior to death, the animals were hypoactive and exhibited irregular respiration and/or prone posture.  Following exposure, one surviving animal was hypoactive and exhibited irregular respiration.  However, the animal recovered by Day 4 and appeared active and healthy for the remainder of the study and gained body weight over the 14-day observation period.  Gross necropsy of the decedents revealed discoloration of the lungs and/or liquid in the trachea. No gross abnormalities were noted for the euthanized animal when necropsied at the conclusion of the 14-day observation period.

In group 4, all animals survived exposure to the test atmosphere. Following exposure, all rats exhibited irregular respiration.  However, all animals recovered by Day 3 and appeared active and healthy for the remainder of the 14-day observation period and gained body weight during the study. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.