5H-Cyclopenta[h]quinazoline, 6,7,8,9-tetrahydro-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:

20 Mar 2014 to 08 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Test material

Test material information

1

Reference substance name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

EC Number:

801-093-8

Cas Number:

1315251-11-6

Molecular formula:

C16H22N2

IUPAC Name:

(8S)-7,7,8,9,9-pentamethyl-5H,6H,7H,8H,9H-cyclopenta[h]quinazoline

Test material form:

solid

Test animals

Species:

rat

Strain:

other: RccHan™ : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Females nulliparous and non-pregnant: yes
- Rationale for use of males (if applicable)
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 200g - 350g
- Housing: Groups of up to three 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"
- Diet: Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK, ad libitum (exception of the exposure period)
- Water: ad libitum (exception of the exposure period)
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 20 Mar 2014 to 08 May 2014

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

7.04 µm

Geometric standard deviation (GSD):

2.59

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany)
- Exposure chamber volume: 30 L (dimensions: 28 cm diameter x 50 cm high)
- Exposure chamber concentration: The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fibre filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals' breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump. Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration. The nominal chamber concentration was calculated by dividing the mass of the substance used by the total volume of air passed through the chamber. The nominal concentration was 1084 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was extremely difficult at what was considered to be the maximum attainable atmosphere concentration.
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During the exposure period, 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.
- Airflow: 30 L/min
- System of generating particulates/aerosols: The substance feed rate from the SAG 410 was maintained at 100 % during the exposure period in an attempt to maintain the test atmosphere at the maximum attainable atmosphere concentration. The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system.
- Method of particle size determination: 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. This device consisted of six impactor stages (8.9, 6.2, 3.6, 1.6, 0.93 and 0.37 µm cut points) with stainless steel collection substrates and a back up 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.
- Temperature: 20 °C
- Humidity: 33-35 %

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Gravimetric method

Duration of exposure:

4 h

Concentrations:

5.02 mg/L

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 were recorded on arrival, prior to treatment on the day of exposure and on days 1, 3, 7 and 14 or at death. 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 fourteen days. Any deaths or evidence of overt toxicity were recorded at each observation.
- Necropsy performed: yes, at the end of the fourteen day observation period the surviving 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.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.02 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

One rat (female) died in a group of ten rats

Clinical signs:

other: Increased respiratory rate, hunched posture, piloerection and wet fur

Remarks:

Frequent instances of tip-toe gait and occasional instances of ataxia and red/brown staining around the snout. Decreased respiratory rate, labored respiration, dehydration, diuresis, dehydration, ptosis and occasional body tremors were also noted.

Body weight:

All animals exhibited body weight losses on the first day post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. In contrast, all surviving female animals exhibited further body weight losses or showed no body weight gains from days 1 to 3 post-exposure. The surviving animals then exhibited body weight gains during the remainder of the recovery period.

Gross pathology:

With the exception of one instance of dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period. Macroscopic abnormalities were noted at necropsy in the animal that died during the course of the study. Dark patches were determined in the lungs and patchy pallor in the liver. Due to the clinical observations noted and macroscopic abnormalities detected, the death noted during the study may have been attributable to systemic toxicity.

Any other information on results incl. tables

Table 1. Mortality data

Mean Maximum Attainable 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
5.02	Male	0	0	0	0	0	0	0	0	0	0	1/10
	Female	0	0	0	0	0	1	0	0	0	0

Table 2. Individual body weights

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Animal Number and Sex	Body Weight (g) on Day:							Increment (g) During Days:
		-8	0	1	3	7	14	At Death	-8-0	0-1	1-3	3-7	7-14
5.02	1 Male	242	294	283	301	312	339		52	-11	18	11	27
	2 Male	232	272	267	278	293	313		40	-5	11	15	20
	3 Male	225	270	259	273	288	316		45	-11	14	15	28
	4 Male	235	274	268	284	293	316		39	-6	16	9	23
	5 Male	227	263	259	268	281	301		36	-4	9	13	20
	6 Female	200	214	193	186	219	234		14	-21	-7	33	15
	7 Female	210	229	208	198	232	239		19	-21	-10	34	7
	8 Female	207	224	213	213	225	228		17	-11	0	12	3
	9 Female	204	222	199	183	-	-	173	18	-23	-	-	-
	10 Female	197	220	205	201	220	233		23	-15	-4	19	13

-  = Animal dead

Applicant's summary and conclusion

Interpretation of results:

other: not harmful in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance has an LC50 > 5.02 mg/L in an OECD TG 403 test.

Executive summary:

A study was performed to assess the acute inhalation toxicity of the substance. The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals No. 403 "Acute Inhalation Toxicity" (2009) and Method B2 (Inhalation) of Commission Regulation (EC) No. 440/2008. A group of 10 RccHan™: WIST strain rats (5 males and 5 females) was exposed to a dust atmosphere. The animals were exposed for 4 hours using a nose only exposure system, followed by a 14 day observation period.

The particulate concentration achieved was measured gravimetrically and found to be 5.02 mg/L The test atmosphere had a mass median aerodynamic diameter of 7.04 µm and a geometric standard deviation (GSD) of 2.59; the inhalable content (%<4 µm) was 27.5%.

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pile-erection and wet fur. There were frequent instances of tip-toe gait and occasional instances of ataxia and red/brown staining around the snout. Isolated occurrences of decreased respiratory rate, laboured respiration, dehydration, diuresis, dehydration, ptosis and occasional body tremors were also noted. The surviving animals recovered to appear normal from days 6 to 11 post-exposure. All animals exhibited body weight losses on the first day post-exposure. All male animals exhibited body weight gains during the remainder of the recovery period. In contrast, all surviving female animals exhibited further body weight losses or showed no body weight gains from days 1 to 3 post-exposure. The surviving animals then exhibited body weight gains during the remainder of the recovery period. With the exception of one instance of dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period.

Based on these results, the LC50 is determined to be greater than 5.02 mg/L.