3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

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 17 March 2016 and 03 May 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 403 in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

acute toxic class method

Limit test:

no

Test material

Test material information

1

Reference substance name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

EC Number:

814-345-7

Cas Number:

2003244-43-5

Molecular formula:

C14H21N

IUPAC Name:

3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Details on test material:

- Name of test material (as cited in study report): ES421 Pinyl Nitrile- Molecular weight (if other than submission substance): 203 g/mol- Physical state: Crystallized white powder- Analytical purity: 98.08% (per Certificate of Analysis)- Composition of test material, percentage of components: 2-Norpinene-2-propionitrile, alpha, alpha,6,6-tetramethyl- Purity test date: 08 February 2016- Lot/batch No.: SM15077102- Storage condition of test material: 2-8°C, protected from light

Specific details on test material used for the study:

Identification: ES421 Pinyl Nitrile

SOURCE OF TEST MATERIAL
- Appearance/physical state: white solid
- Source and lot/batch No.of test material: SM15077102
- Expiration date of the lot/batch: 22 July 2016
- Purity: 98.08%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately 4 °C, in the dark

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 (ES421 Pinyl Nitrile : Ethanol, 50 : 50 w/w) to improve the aerosolization properties of the test item. Prior to each use, the test item was warmed in a warming bath (set at 80 °C) in order to liquefy the test item. During exposure, the test item formulation was kept in a warming bath set at 80 °C to ensure the test item didn’t solidify.

Test animals

Species:

rat

Strain:

other: RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
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 5 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 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.

Animal Care and Husbandry
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. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would 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 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Atmosphere Generation
The test item formulation was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, 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 under pressure, and to a metered compressed air supply.
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.
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 considered to be the maximum attainable as the syringe pump was left on the maximum pump rate for the duration of the exposure period. 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. A diagram of the dynamic (continuous flow) system employed is shown in Figure 1.
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, extensive work was performed in an attempt to achieve a sustainable atmosphere at maximum concentration. This included varying test item formulation input rates, air flow settings and generation systems. No significant improvement could be made to the achieved atmosphere concentrations and, therefore, the concentration achieved is considered the maximum attainable.

Exposure Procedure
One day 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.
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 (ES421 Pinyl Nitrile : Ethanol, 50 : 50 w/w) for a period of four hours. A target concentration of 5 mg/L was used for the exposure but, due to the poor aerosolization properties of the test item, this could not be achieved. The animals were therefore exposed to the maximum attainable concentration. No deaths occurred and therefore 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 30 minutes throughout the 4 Hour exposure period. Individual values are shown under 'Any other information on materials and methods incl. tables'.

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 4 Hour exposure period. The test atmosphere was generated to contain at least 19% oxygen. Individual values are shown under 'Any other information on materials and methods incl. tables'.

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 66.16 % (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. The 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 843% of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was extremely difficult, even 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.

References
Green, J.D. et al (1984) Effect of Equilibration Zones on Stability, Uniformity and Homogeneity Profiles of Vapours and Aerosols in the ADG Nose Only Inhalation Exposure System. Fundamental and Applied Toxicology 4, 768-777.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Mean maximum attainable atmosphere concentration: 4.28 mg/L

No. of animals per sex per dose:

Five per sex per dose

Control animals:

no

Details on study design:

Serial 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, 1 hour after termination of exposure and subsequently once daily for 14 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 (Day 0) and on Days 1, 3, 7 and 14.

Terminal Investigations

Necropsy
At the end of the 14 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 forsigns of irritancy or local toxicity.

Data Evaluation
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.

The following computerized system was used in the study: Delta Controls – ORCAview (Version 3.4.0)

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.28 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No deaths occurred in a group of ten rats exposed to a mean maximum attainable atmosphere concentration of 4.28 mg/L for 4 hours. For details refer to 'Any other information on results incl. tables'.

Clinical signs:

other: Common abnormalities noted during the study included decreased respiratory rate, noisy respiration, sneezing, hunched posture, pilo-erection and wet fur. Isolated instances of labored respiration and red/brown staining around the snout were also noted. An

Body weight:

All males and four female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. With the exception of two female animals which showed body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period. For details refer to 'Any other information on results incl. tables'.

Gross pathology:

No macroscopic abnormalities were detected amongst animals at necropsy. Terminal kill day 14.

Other findings:

Exposure Chamber Concentration
The test atmosphere was sampled seventeen times during the exposure period and the actual concentration of the test item calculated. For details refer to 'Any other information on results incl. tables'.

Particle Size Distribution
The particle size analysis of the atmosphere drawn from the animals’ breathing zone is summarized under 'Any other information on results incl. tables'.
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* (Silver, 1946).

References
Silver, S.D. (1946) Constant flow gassing chambers: Principles influencing design and operation. J Lab Clin Med 31, 1153-1161.

Any other information on results incl. tables

Mortality

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Sex	Deaths		Deaths Noted During Period of Observation (Days)								Total Deaths
		During Exposure Period	1 Hour Post Exposure	1	2	3	4	5	6	7	8-14
4.28	Male	0	0	0	0	0	0	0	0	0	0	0/10
	Female	0	0	0	0	0	0	0	0	0	0

Individual Clinical Observations – (Day of Exposure)

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Animal Number and Sex	Effects Noted During Exposure (Hours)			Effects Noted on Removal from Chamber	Effects Noted 1 Hour Post Exposure
		1	2	3
4.28	1 Male	WfRd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	2 Male	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	3 Male	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	4 Male	Wf Rd	Wf Rd	Wf Rd Rl	Wf H P Rd Rn	Wf H P Rd Rn
	5 Male	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	6 Female	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	7 Female	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	8 Female	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn Ss	Wf H P Rd Rn
	9 Female	Wf Rd	Wf Rd	Wf Rd	Wf H P Rd Rn	Wf H P Rd Rn
	10 Female	Wf Rd	Wf Rd Rl	Wf Rd Rl	Wf H P Rd Rn	Wf H P Rd Rn

Wf= Wet Fur; Rd = Decreased respiratory rate; Rl = labored respiration; Rn = Noisy respiration; H = Hunched posture; P = Pilo-erection; Ss = red/brown staining around the snout

Individual Clinical Observations – Recovery Period

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Animal Number and Sex	Effects Noted Post Exposure (Days)
		1	2	3	4	5	6	7	8-14
4.28	1 Male	HRs	0	0	0	0	0	0	0
	2 Male	H Rs	0	0	0	0	0	0	0
	3 Male	H Rs	0	0	0	0	0	0	0
	4 Male	H Rs	0	0	0	0	0	0	0
	5 Male	H Rs	0	0	0	0	0	0	0
	6 Female	H Rs	0	0	0	0	0	0	0
	7 Female	H Rs	0	0	0	0	0	0	0
	8 Female	H Rs	0	0	0	0	0	0	0
	9 Female	H Rs	0	0	0	0	0	0	0
	10 Female	H Rs	0	0	0	0	0	0	0

H= Hunched posture; Rs = sneezing; 0 = No abnormalities detected

Individual Body Weights and Body Weight Changes

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Animal Number and Sex	Body Weight (g) at Day						Body Weight Change (g) During Days
		-27	0	1	3	7	14	-27 to 0	0 to 1	1 to 3	3 to 7	7 to 14
4.28	1 Male	194	331	331	337	349	372	137	0	6	12	23
	2 Male	193	321	316	331	347	368	128	-5	15	16	21
	3 Male	203	328	325	333	341	378	125	-3	8	8	37
	4 Male	203	336	336	344	356	384	133	0	8	12	28
	5 Male	190	280	269	271	286	311	90	-11	2	15	25
	6 Female	195	228	225	229	236	242	33	-3	4	7	6
	7 Female	193	228	224	233	236	249	35	-4	9	3	13
	8 Female	198	234	233	229	239	245	36	-1	-4	10	6
	9 Female	190	219	219	221	229	238	29	0	2	8	9
	10 Female	190	227	229	225	229	235	37	2	-4	4	6

Exposure Chamber Concentration

Duration of Exposure (minutes)	Amount of Non-Volatile Component* Collected During Sample (mg)	Equivalent Quantity of Test Item (mg)	Volume of Air Sampled (L)	Atmosphere Concentration(mg/L)
5	5.08	7.68	2	3.84
15	5.31	8.03	2	4.01
30	6.14	9.28	2	4.64
45	4.98	7.53	2	3.76
60	6.79	10.26	2	5.13
75	5.19	7.84	2	3.92
90	4.49	6.79	2	3.39
105	4.74	7.16	2	3.58
120	5.17	7.81	2	3.91
135	5.20	7.86	2	3.93
150	5.97	9.02	2	4.51
165	6.44	9.73	2	4.87
180	6.29	9.51	2	4.75
195	5.21	7.87	2	3.94
210	7.11	10.75	2	5.37
225	7.65	11.56	2	5.78
235	4.62	6.98	2	3.49

Mean maximum attainable atmosphere concentration (mg/L)		=4.28
Standard deviation		=0.70
Nominal concentration:
Test item used (g)	543
Air flow (L/minute)	60
Total generation time (minutes)	251**
Nominal concentration (mg/L)	36.1

*Non-volatile component = 66.16 % of ES421 Pinyl Nitrile

**Test atmospheres were generated for a total of 11 minutes prior to animal insertion to ensure test item concentration was being achieved.

Particle Size Distribution

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
4.28	2.94	63.8	2.40

Applicant's summary and conclusion

Conclusions:

No deaths occurred in a group of ten rats exposed to a mean maximum attainable atmosphere concentration of 4.28 mg/L for 4 hours. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item ES421 Pinyl Nitrile in the Wistar strain rat was greater than 4.28 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute inhalation toxicity of the test item ES421 Pinyl Nitrile. The method used was designed to be compatible with that described in the OECD Guideline for Testing of Chemicals No. 403 “Acute Inhalation Toxicity” (2009) and Method B.2. (Inhalation) of Commission Regulation (EC) No. 440/2008.

Methods

A group often RccHan^TM: WIST strain rats (five males and five females)was exposed to an aerosol atmosphere of a formulation of the test item (ES421 PinylNitrile: Ethanol, 50 : 50 w/w). The animals were exposed for 4 hours using a nose only exposure system, followed by a fourteen day observation period.

Results

The mean maximum attainable atmosphere concentration was as follows:

Atmosphere Concentration
Mean Maximum Attainable (mg/L)	Standard Deviation	Nominal (mg/L)
4.28	0.70	36.1

The characteristics of the achieved atmosphere were as follows:

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Mean Mass Median Aerodynamic Diameter (µm)	Inhalable Fraction (% <4 µm)	Geometric Standard Deviation
4.28	2.94	63.8	2.40

The mortality data were summarized as follows:

Mean Maximum Attainable Atmosphere Concentration (mg/L)	Deaths
	Male	Female	Total
4.28	0/5	0/5	0/10

Clinical Observations: Common abnormalities noted during the study included decreased respiratory rate, noisy respiration, sneezing, hunched posture, pilo-erection and wet fur. Isolated instances of labored respiration and red/brown staining around the snoutwere also noted. Animals recovered to appear normal on Day 2 post-exposure.

Body Weight: All males and four female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. With the exception of two female animals which showed body weight losses from Days 1 to 3 post-exposure, body weight gains were noted for all animals during the remainder of the recovery period.   

 

Necropsy: No macroscopic abnormalities were detected amongst animals at necropsy.