Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods, therefore it is considered relevant, reliable and adequate for classification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-Methyl-4-(1-methylethenyl)cyclohexene and 1-Methyl-4-(1-methylethylidene)-cyclohexene and 1-methyl-4-(propan-2-yl)cyclohexa-1,3-diene

EC Number:

939-409-2

Cas Number:

1329-99-3

Molecular formula:

C10H16

IUPAC Name:

Reaction mass of 1-Methyl-4-(1-methylethenyl)cyclohexene and 1-Methyl-4-(1-methylethylidene)-cyclohexene and 1-methyl-4-(propan-2-yl)cyclohexa-1,3-diene

Constituent 2

Reference substance name:

Isopropylmethylcyclohexane, tetradehydro derivative

EC Number:

215-532-0

EC Name:

Isopropylmethylcyclohexane, tetradehydro derivative

Cas Number:

1329-99-3

IUPAC Name:

1-isopropyl-4-methylcyclohexane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Depanol I; isopropylmethylcyclohexane, tetradehydro derivative; p-mentadiene
- Substance type: Organic multi constituent
- Physical state: Liquid, colourless, characteristic odour
- Analytical purity: Terpene hydrocarbons: 98.9% (w/w); p- Menthadienes: 96.3 (w/w)
- Composition of test material, percentage of components & impurities: See confidential details
- Purity test date: 06-07-2011
- Lot/batch No.: DEFM102174
- Expiration date of the lot/batch: 25-06-2013
- Stability under test conditions: Not provided
- Storage condition of test material: Ambient (+18 to +36°C)

Test animals

Species:

rat

Strain:

other: Wistar rats- HsdCpb:WU

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, India
- Age at study initiation: At exposure: 10-11 weeks
- Weight at study initiation: At grouping: Males: 270.83 – 285.26 g; Females: 212.92 – 225.78 g
At start of treatment: Males: 273.28 – 287.01 g; Females: 214.86 – 227.77 g
- Fasting period before study: Food and water were withheld during the exposure period.
- Housing:Individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175mm), with stainless steel top grill having facilities for pelletted food and drinking water; Bedding: steam sterilized corn cob was used and changed along with the cage twice a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet- Pellet (Certified) manufactured by Harlan Laboratories B.V. Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 65-68%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2-10-2012 To: 24-10-2012

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: Milli-Q water

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass atomizer; injection rate of 0.4 mL/min with 1.4 kg/cm2 of atomizer pressure.
The equipment, nose only exposure chamber is made of two stainless steel cylindrical chambers with 24 exposure ports in 4 tiers (6 ports per tier) to house up to 24 animals. The equipment consists of two chambers, i.e. the aerosol inlet chamber and the aerosol exhaust chamber. The chambers are cylindrical in shape and placed one next to the other. The chamber has 24 ports into a central plenum arranged as 4 horizontal rows centered on the sides of the cylindrical chamber. Within the plenum is a central tube into which the aerosol enters at each port. Air flow carries the aerosol to the nose of the animals restrained in position at the open ends of the tubes. The plexiglass restrainers with the animals are housed in an outer chamber.This chamber is made up of cylindrical plexiglass cover with an opening to fix the restrainers to the central plenum. The chamber has temperature and humidity control devices, and the temperature and the humidity where the animals are housed can be controlled and monitored.
- Exposure chamber volume: The approximate rat restrainer size for males: length 19.0 cm, diameter 6.0 cm and for females: length 15.0 cm, diameter 6.0 cm.
- Method of holding animals in test chamber: The animal restrainers are made of polycarbonate tubes for individual exposure tubes.
- Source and rate of air: The airflow rate was monitored continuously and the airflow of 20 L/min was maintained.
- Method of conditioning air: Aerosol not breathed and expired air pass into the plenum chamber (aerosol exhaust chamber) and from there, via filters, to the exhaust system.
- System of generating particulates/aerosols: A glass atomizer was used.
- Method of particle size determination: GALAI CIS-50 particle size analyzer; Laser based ‘Time-of-Transition Theory’;
- Treatment of exhaust air: Passed via filters to the exhaust system.
- Temperature: Inner chamber: 20.0-21.2 °C; Outer chamber: 20.3-21.1 °C.
Humidity: Inner chamber: 64.45 - 67.23 °C; Outer chamber: 64.85 - 67.65 °C.

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples were drawn from one port at hourly intervals of the 4 hour exposure period. The chamber air was passed through the sampling column which contains the silica gel (60–120 mesh size: approximately 16-20 gm) by suction applied using vacuum pump at a rate of 10 L/min for 10 minutes. The test item concentration in the air inhalation sample columns were analyzed using a validated analytical method.
The test item concentration in the air inhalation sample columns were analyzed using a validated analytical method.
The inhalation columns were eluted with Ethyl acetate. The concentration of Depanol I was determined by using Gas Chromatograph (GC) equipped with FID detector, auto sampler and a PC based data system.
- Samples taken from breathing zone: Yes.

VEHICLE
- Composition of vehicle (if applicable): Milli-Q water

TEST ATMOSPHERE
- Particle size distribution:The aerosol particle size was analyzed thrice using GALAI-CIS-50 particle size analyzer, i.e., during the first, second and fourth hour of the exposure period. The air samples from the chamber were passed through the cuvette of the aerosol module of the instrument which measured the aerosol particle size by laser based “Time of transition theory” and instantaneous data on mean particle size with standard deviation were obtained from the computer and the results were as follows.

Trial Number Sampling hour Particle size in micrometer
Mean ± SD
1 I 1.26 ± 0.95
2 II 1.27 ± 0.98
3 IV 1.20 ± 0.88
Overall mean ± SD 1.21 ± 0.98
Calculated Geometric standard deviation 2.46

- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The value for geometric standard deviation (GSD) for the test item is 2.46. This indicates that the size distribution of the aerosols is acceptable (GSD falls within the range of 1.5-3.0, as recommended by OECD guidance document no. 39 on acute inhalation testing).

Analytical verification of test atmosphere concentrations:

yes

Remarks:

The inhalation columns were eluted with Ethyl acetate. The concentration of Depanol I was determined by using Gas Chromatograph (GC) equipped with FID detector, auto sampler and a PC based data system

Duration of exposure:

4 h

Concentrations:

Analyzed Concentration, mg of test item/L of chamber air
Mean ± SD: 4.95 ± 0.472

No. of animals per sex per dose:

5 (Main study)
3 (Pre-study)

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Toxic signs could not be ascertained four times at hourly intervals during the exposure period due to the special housing method. The observations for toxic signs and pre-terminal deaths were done immediately after exposure, while releasing the rats from the restrainers. The rats were observed twice after release of rats from the restrainers. Thereafter, the observations for toxic signs and pre-terminal deaths were done once daily during days 2 to 15.
Body weights were recorded once during the acclimatization period and on day 1 (pre-exposure), 2, 4, 8 (7 days of post exposure) and 15 (14 days of post exposure.
- Necropsy of survivors performed: Yes.

Results and discussion

Preliminary study:

A pre-study was conducted as per OECD 403 Traditional Protocol using 3 male and 3 female rats, the rats were exposed to the test item aerosol generated under the following conditions:

Test item concentration Undiluted test item
Atomizer used Glass
Atomizer Pressure 1.4 kg/cm2
Injection rate 0.4 mL/min.
Airflow rate 20 L/ min.
Sampling duration 10 min.
Sampling rate 10 L/min.

The suspendability / solubility test was not performed since the undiluted test item was used with an injection rate of 0.4 mL / min for the pre-study.
The animals were exposed in inhalation equipment with dynamic airflow.
The results of pre-study are given below.
G1 Undiluted test item 3 M/3F

1) Test item concentration# inChamber air, mg/L : 6.52 ± 1.06
2) Aerosol particle size (µm): 1.50 ± .1.34

*: Toxic signs could not be ascertained during the inhalation exposure period of four hours due to special housing method and the toxic signs recorded were at the time of release of animals from the restrainers. Tremors, slight salivation and nasal discharge were observed in two male and two female. Tremors and nasal discharge were observed in one male and one female on day1. The rats were normal from day2.
#: The test item concentration in the air inhalation sample columns was analysed using a validated analytical method.
The necropsy findings will be recorded in the raw data and included in the report.
Based on the pre-study result, the following dose is selected for the main study.
G1: Undiluted test item was used with an injection rate of 0.4 mL/min.

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.95 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: nose only

Mortality:

There were no mortalities.

Clinical signs:

other: The clinical signs of toxicity were nasal discharge, slight ataxia, slight salivation and slight tremors immediately and approximately 1 hour after end of exposure on day 1 and all the rats were normal form day 2 onwards.

Body weight:

There was a slight decrease in body weight of five female and three male rats on day 2 when compared to their initial body weight. On day 4 there was a slight decrease in body weight of three female rats when compared to their initial body weight, however all the rats gained weight during 8th and 15th day weighing.

Gross pathology:

No abnormality was detected at necropsy in any of the rats.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute inhalation (4 hours) Lethal Concentration (LC50) value of Depanol I in Wistar rats is more than 4.95 mg/L of chamber air (maximum attainable concentration) in both male and female Wistar rats.

Executive summary:

The inhalation toxicity of Depanol I was determined in 5 male and 5 female Wistar rats by exposure to aerosol of undiluted test item generated by glass atomizer with an injection rate of 0.4 mL/min. with 1.4 kg/cm2 of atomizer pressure. The rats were housed in special rat restrainers and were continuously exposed to the test item aerosol (nose only) for four hours in inhalation exposure chamber (dynamic state). The post treatment observation period was for 14 days. The aerosol sampled from the inhalation chamber for particle size analysis showed a mean aerosol particle size of 1.21 ± 0.98 micrometers. The generated aerosol particle distribution was uniform throughout the exposure period of four hours. The geometric standard deviation (GSD) value of the test item was 2.46, indicating that the aerosol was polydisperse. The fraction of aerosol with particle size of 3.90 micrometer was less than 97% and this fraction was considered to be inhalable. The analytically determined average concentration of Depanol I was 4.95 mg/L of chamber air. There were no pre-terminal deaths. The clinical signs of toxicity were nasal discharge, slight ataxia, slight salivation and slight tremors immediately and approximately 1 hour after end of exposure (Day1). The rats were normal from day 2 onwards.

The acute inhalation (4 hours) Lethal Concentration (LC50) value of Depanol I was more than 4.95 mg/L of chamber air (maximum attainable concentration) in both male and female Wistar rats. Based on the test results of the present study, the test item, Depanol I is not classified.