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

Endpoint summary

Description of key information

Key studies were conducted with Depanol I for acute oral, inhalation and dermal toxicity. In all studies, LD50 or LC50 values were above limit dose, therefore Depanol I is safe and does not have any acute hazard potential. 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Wistar – HsdCpb: WU

Sex:

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: 9 to 10 weeks at treatment
- Weight at study initiation: 185.40 – 200.17 g
- Fasting period before study: Yes, 16-18 hours; water was not withheld. Food was offered 3-4 hours after dosing.
- Housing: Individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities for pelletted food and drinking water. Bedding: steam sterilized clean paddy husk 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 Netherland; ad libitum.
- 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, in polycarbonate bottles with stainless steel sipper tubes; ad libitum.
- Acclimation period: From 5 to 7 days under laboratory condition after physical examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 58-67%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9, 2012 To: August 30, 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage):
- Justification for choice of vehicle: A quantity of 1.0 g of the test item was mixed with Milli-Q water and the volume made up to 5 mL. The test item was not miscible.
Hence a quantity of 1 g of test item was mixed with corn oil and the volume was made up to 5 mL to get the test item concentration of 200 mg/mL suspension. The test item formed a visibly homogenous suspension. Hence, corn oil was used as the vehicle to prepare the test item suspension.
- Lot/batch no. (if required): MKBG9425V

DOSAGE PREPARATION (if unusual): The test item suspension was prepared on each day of the test item administration. A quantity of 4.0 g of the test item was mixed by adding small quantity of the selected vehicle (corn oil) and the volume made up to 20 mL to get the test item concentration of 200 mg/mL suspension. Homogeneity of the test item in the vehicle was maintained by mixing, using glass rod, prior to treatment. Preparation was made prior (within 1 hour) to dosing.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: As per the Material Safety Data Sheet provided by the Sponsor, the Acute Oral toxicity (mg/kg) in mouse is >2000 mg/kg body weight, hence the test was started as per Annex 2d of the OECD 423. The starting dose was 2000 mg/kg body weight with 3 animals /step.

Doses:

2000mg/kg bw

No. of animals per sex per dose:

3 females per treatment step

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations: 5x on test day 1 and 1x daily during days 2-15 post administration; Weighing: on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration).
- Necropsy of survivors performed: yes

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: as per LD50 cut-off value

Mortality:

No pre-terminal deaths.

Clinical signs:

other: No clinical signs.

Gross pathology:

No abnormality was detected at necropsy in control and treatment groups.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Depanol I has an oral LD50 of >2000 mg/kg body weight and does not have to be classified as per CLP Regulation.

Executive summary:

The acute oral toxicity study with Depanol I in Wistar rats was tested according to the acute toxic class method. The test item suspended in corn oil was administered as oral gavage to overnight fasted (16 – 18 hours) 3 female rats at the dose of 2000 mg/kg body weight. Vehicle control group animals were administered corn oil. There were no toxic signs and pre-terminal deaths in treatment and control groups. Three additional female rats were tested at the same dose of 2000 mg/kg body weight. There were no toxic signs and pre-terminal deaths in treated and control groups. All animals of both groups gained body weight during 14 days observation period.

The rats of treatment and control groups were subjected to necropsy at termination and there were no abnormalities detected.

Based on the results of the present study, the test item, Depanol I has an oral LD50 of >2000 mg/kg body weight and does not have to be classified as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

High quality study

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

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

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

4.95 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

A key oral toxicity study with Depanol I in Wistar rats was conducted according to the acute toxic class method (Advinus, 2012a). The test item suspended in corn oil was administered as oral gavage to overnight fasted 6 female rats at the dose of 2000 mg/kg body weight, whereas vehicle control group animals were administered corn oil. There were no toxic signs and pre-terminal deaths in treatment and control groups. All animals gained body weight during 14 days observation period. Necropsy at termination did not reveal abnormalities. Based on the results of the present study, the test item, Depanol I has an oral LD50 of >2000 mg/kg body weight and does not have to be classified as per CLP Regulation. In a supporting study, the LD50 of Depanol I in three solvents (Pinene-Camphor,Terpin hydrate and Flotol) was determined in mice after single oral exposure. The LD50 of Depanol I in Pinene-Camphor was 5000 mg/kg bw, in Terpin hydrate 6000 mg/kg bw and in Flotol 2000 mg/kg bw. It was concluded that the test item was not particularly toxic (relatively harmless).

A key acute inhalation toxicity with Depanol I was conducted 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 (Advinus, 2012b). 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 toxic signs and pre-terminal deaths. No abnormality was detected in any of the rats. The acute inhalation (4 hours) 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.

A key acute dermal toxicity test was conducted with Wistar rats (male and female) to determine the potential for Depanol I to produce toxicity from a short term exposure via the dermal route (Advinus, 2012c). The undiluted test item at the dose of 2000 mg/kg bw was applied directly to the clipped skin of the animal to cover about 10% of body surface under adhesive occlusion for 24 hours. After the 24 hour contact period, the dressing was removed and the applied area was washed initially with water and with Johnson’s Baby Soap and again with water. Washed animals were wiped dry with a cotton hand towel.  After treatment in 5 female rats, 5 male rats were treated. All the rats were observed for clinical signs of toxicity, local skin reaction and mortality for 14 days post application. There were no clinical signs of toxicity, local skin reaction or mortality. There were no abnormalities detected at the necropsy. Based on the results of the present study, the LD50 was above 2000 mg/kg bw and the test item does not have to be classified.

Justification for selection of acute toxicity – oral endpoint
Key study with Depanol I

Justification for selection of acute toxicity – inhalation endpoint
Key study

Justification for classification or non-classification

Based on the available data, Depanol I does not have to be classified for acute toxicity according the EU labelling regulations Commission Directive 93/21/EEC or CLP regulation No. 1272/2008 of 16 December 2008.