5H-Cyclopenta[h]quinazoline, 6,6a,7,8,9,9a-hexahydro-7,7,8,9,9-pentamethyl-

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 02 July 2015 and 11 August 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

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:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

EC Number:

801-694-5

Cas Number:

1392325-86-8

Molecular formula:

C16H24N2

IUPAC Name:

7,7,8,9,9-pentamethyl-5H,6H,6aH,7H,8H,9H,9aH-cyclopenta[h]quinazoline

Test material form:

other: Solid

Details on test material:

Identification: IFF TM 12-206 (FRET 10-0199)
Description: White solid block
Storage conditions: Approximately 4 °C, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately eight to twelve weeks old and within the weight range of 190g to 350g. The females were nulliparous and non-pregnant.

The animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK). With the exception of the exposure period, free access to mains drinking water and food (Harlan 2014C Rodent Diet, Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding, chew blocks and cardboard “fun tunnels” are routinely analyzed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

The nature of the test item was such that a suitable atmosphere could not be generated from the test item as supplied. A formulation was, therefore, prepared with ethanol, (IFF TM 12-206 (Fret 10-0199) : ethanol, 50 : 50 w/w) to improve the aerosolization properties of the test item.

Atmosphere Generation
The test item formulation was aerosolized using a metal concentric jet nebulizer (HLS, UK) located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item formulation under pressure, and to a metered compressed air supply. The tubing which contained the test item prior to being introduced to the nebulizer was passed through a warming bath maintained at approximately 75°C in order to prevent the test item formulation solidifying during the generation process.

Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the nebulizer. This airline was also passed through a warming bath maintained at approximately 75°C in order to prevent the test item formulation solidifying during the generation process.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.

Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period, test item formulation input rates, air flow settings and the generation system were varied in an attempt to achieve the required atmospheric conditions.

Exposure Procedure
One day prior to exposure, each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the day of exposure, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.

Following an appropriate equilibration period, a single group of ten rats (five males and five females) was exposed to an atmosphere of a formulation of the test item (IFF TM 12-206 (Fret 10-0199) : ethanol, 50 : 50 w/w) for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure, as the mean achieved concentration was 102 % of target and no deaths occurred, no further levels were required.

Exposure Chamber Temperature and Relative Humidity
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period.

Exposure Chamber Oxygen Concentration
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

Exposure Chamber Atmosphere Concentration
Prior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fiber filters and recording their weights. The filters were then dried in a desiccator at room temperature for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the formal exposure was found to be 98.28 % (n=10).

The test atmosphere was sampled at regular intervals during the exposure period. A weighed glass fibre filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump.

After sampling, the filter was dried, under similar conditions as those previously described, and weighed again 24 hours later. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was the chamber concentration in terms of non-volatile component. Additional ethanol added to improve aerosolization was considered not to affect the gravimetric calculation as all of the filters were dried thoroughly (for 24 hours) prior to weighing.

Based on the results of the preliminary work, these figures were adjusted to obtain a true figure for the test item concentration in the chamber.

The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.

The nominal concentration was 306 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward when formulating the test item with ethanol.

Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.

The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.

The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 µm was calculated.

The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5.11 mg/L.

No. of animals per sex per dose:

Five per sex per dose

Control animals:

no

Details on study design:

Observations
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of overt toxicity was recorded at each observation.


Body Weight
Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.


Necropsy
At the end of the fourteen day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.


Evaluation of Data
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.

Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.11 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There were no deaths at the mean achieved atmosphere concentration of 5.11 mg/L.

Clinical signs:

other:

Body weight:

All animals exhibited body weight losses on the first day post-exposure. With the exception of one female animal which showed no body weight gain from Days 3 to 7 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period.

Gross pathology:

Dark patches on the lungs were noted at necropsy of three animals. No macroscopic abnormalities were detected amongst the other seven animals.

Any other information on results incl. tables

Exposure Chamber Concentration

The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were:

 

Atmosphere Concentration
Mean Achieved (mg/L)	Standard Deviation	Nominal (mg/L)
5.11	0.13	15.6

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

The theoretical chamber equilibration time (T₉₉) was 3 minutes[1] (Silver, 1946).

[1] = Test atmospheres were generated for a total of 78 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone, was as follows:

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
5.11	1.77	86.9	2.08

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)	Amount of Non-Volatile Component[1]Collected During Sample (mg)	Equivalent Quantity of Test Item (mg)	Volume of Air Sampled (L)	Atmosphere Concentration(mg/L)
5	9.93	10.10	2	5.05
15	9.99	10.16	2	5.08
30	9.89	10.06	2	5.03
45	9.82	9.99	2	5.00
60	10.13	10.31	2	5.15
75	10.03	10.21	2	5.10
90	10.06	10.24	2	5.12
105	9.73	9.90	2	4.95
120	10.05	10.23	2	5.11
135	10.83	11.02	2	5.51
150	9.83	10.00	2	5.00
165	10.19	10.37	2	5.18
180	10.24	10.42	2	5.21
195	10.01	10.19	2	5.09
210	9.98	10.15	2	5.08
225	10.09	10.27	2	5.13
235	9.79	9.96	2	4.98

Meanachievedatmosphere concentration (mg/L) =5.11

Standard deviation =0.13

[1] Non-volatile component = 98.28 % of IFF TM 12-206 (Fret 10-0199)

Particle Size Distribution

Cascade Impactor Data 

Impactor Stage Number	Cut Point (µm)	Amount Collected (mg) per Sample Number			Mean Amount Collected (mg)
		1	2	3
3	8.4	0.02	0.04	0.03	0.03
4	7.3	0.05	0.08	0.05	0.06
5	3.6	0.45	0.51	0.41	0.46
6	1.3	0.68	0.65	0.61	0.65
7	0.94	0.97	1.02	0.93	0.97
8	0.43	0.19	0.18	0.30	0.22
Back-up Filter	<0.43	0.07	0.09	0.07	0.08
Total Mean Amount of Test Item Collected					2.47

 

Calculation

Cut Point (µm)	Log10 Cut Point	Mean Cumulative Amount Less Than Cut Point
		(mg)	(%)	Probit
8.4	0.924	2.44	98.8	7.25
7.3	0.863	2.38	96.4	6.79
3.6	0.556	1.92	77.7	5.76
1.3	0.114	1.27	51.4	5.04
0.94	-0.027	0.30	12.1	3.83
0.43	-0.367	0.08	3.24	3.15

Results

Mean Mass Median Aerodynamic Diameter (MMAD) = 1.77µm

Geometric Standard Deviation (GSD) = 2.08

Predicted amount less than 4 µm = 86.9%

Mortality Data

Mean Achieved Atmosphere Concentration (mg/L)	Sex	Deaths During Exposure	Deaths Post Exposure (1 Hour)	Deaths During Day of Observation								Total Deaths
				1	2	3	4	5	6	7	8-14
5.11	Male	0	0	0	0	0	0	0	0	0	0	0/10
	Female	0	0	0	0	0	0	0	0	0	0

Applicant's summary and conclusion

Interpretation of results:

other: Not harmful

Remarks:

in accordance with EU CLP (EC no. 1272/2008 and its amendments)

Conclusions:

The substance has an LD50 of >5.11 mg/L air in an OECD TG 403 test.

Executive summary:

The substance was tested in an acute inhalation test (OECD TG 403) at a dose level of 5.11 mg/L. No mortality occurred. Clinical signs observed during the study included decreased respiratory rate, noisy respiration, ataxia, hunched posture, pilo-erection and wet fur. Animals recovered to appear normal from days 3 to 4 post-exposure. Macroscopy showed dark patches on the lungs for three animals. No macroscopic abnormalities were detected amongst the other seven animals. The LC50 result in >5.11 mg/L air.