Butanal, 4-(heptyloxy)-3-methyl-

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 13 May 2014 and 02 July 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guidelines 436 using the acute toxic class method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(heptyloxy)-3-methylbutanal

EC Number:

802-100-7

Cas Number:

1093653-57-6

Molecular formula:

C12H24O2

IUPAC Name:

4-(heptyloxy)-3-methylbutanal

Test material form:

other: liquid

Details on test material:

Test substance: TM 09-217
Test substance identity: 4-(heptyloxy)-3-methyl butanal
CAS number: 1093653-57-6
Intended use: Fragrance in household and personal care products.
Description: Liquid
Storage conditions: At ambient temperature (ca. 20 °C) protected from light
Supplier: Sponsor

The Sponsor was responsible for the characterisation of the test substance and the documentation of the methods of synthesis, fabrication or derivation and stability.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Supply, Acclimitisation and allocation
4 male and 4 female Crl:CD (SD) rats were received from a reputable supplier. The rats were ordered at 56 to 63 days of age and within a weight range of ±20% of the mean bodyweight for each sex.

On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Each animal was assigned a number and identified uniquely within the study by a tail mark. Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

The animals were allowed to acclimatise to the conditions described below for 27 days before treatment commenced. For those animals selected for this study, their age at the start of treatment was 83 to 90 days and their body weights were in the range of 450 to 507 g for males and 260 to 286 g for females. The spare animals were removed from the study room after treatment commenced.

Animal Housing, Diet and Water Supply
Animals were housed inside a restricted entry rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms. Before the study the room was cleaned and disinfected. Each animal room was supplied with filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were maintained within the range of 19 to 23°C and 40 to 70% respectively. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Temperature and humidity were monitored continuously. Since these data show that there were no significant deviations from target values they are not presented. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.

The animals were housed four of one sex per cage during the pretreatment phase and three of one sex per cage from the day of exposure (males and females were housed separately). The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood shavings (Lignocel 3/4) were used as bedding and were sterilised by autoclaving and changed at appropriate intervals each week. Cages, food hoppers and water bottles were changed at appropriate intervals.

Whilst in the home cage, animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes. Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages. Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the wood shavings and Aspen chew blocks. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.

No other specific contaminants that were likely to have been present in the wood shavings, chew blocks, diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

not specified

Details on inhalation exposure:

Group 1 received a single 4-hour exposure to TM09-217

Each exposure system comsisted of stainless steel concentric jet atomiser and a snout only exposure chamber.

The inhalation exposure system comprised an ADG snout-only inhalation exposure chamber, restraining tubes, a jet atomiser and extract lines, which attached to the top and bottom of the chamber respectively and an air supply to the atomiser. A filtration system was incorporated in the extract line. The aerosol generator, a stainless steel concentric jet atomiser, was designed to produce and maintain an atmosphere containing a high proportion of respirable droplets.

The test substance was supplied to the generator, via the feed line (Vygon), from a 60 mL syringe (Polypropylene) driven at a constant rate by a syringe pump (Harvard). The ADG snout-only inhalation chamber was a modular apparatus of aluminium alloy construction comprising a base unit, a single animal exposure section with 20 ports and a top section incorporating a central aerosol inlet. The jet atomiser was mounted directly into the central aerosol inlet of the ADG chamber top section. The dimensions of the chamber are 45 cm high with a diameter of 30 cm, with an internal volume of approximately 30.0 litres. During dosing the chamber was housed in its own enclosed ventilated cabinet.

The compressed air supply was filtered to remove any residual particulate and dried. This was used to operate the jet atomiser. Air extract, attached to the base of the inhalation exposure system, was provided by vacuum pumps and incorporated a filtration system to remove test material. The compressed air flow rate exiting the jet atomiser was 11 litres/minute; the extract airflow rate was set at 20 litres/minute. Air supply and extract were monitored using in-line flowmeters, checked at intervals throughout the exposure and recorded manually. The in-line flowmeters were calibrated prior to the exposure against high quality tapered tube rotameters and were used to measure the free flow of air at the points of attachment of the supply and extract lines to the exposure system.

Prior to the start of the study, trials were conducted to establish the inhalation exposure system operating conditions required to generate the target chamber concentration. Initially trials were conducted using a glass vaporiser, however vapour generation was not successful. The generation device was changed to a jet atomiser which was successful in aerosol generation. These results are not reported but are retained in the raw data.

Chamber Aerosol Characterisation
A measured volume of air was drawn at a rate of 2 litres/minute from an unused exposure port on the exposure chamber through a bubbler, using a wet type gas meter. Five samples were collected during the exposure and retained for chemical analysis. Additional filter samples were also performed throughout the exposure for monitoring purposes only. This data is documented in the study data but is not reported.

Droplet Size Distribution
A measured volume of air was drawn at a rate of 2 litres/minute from an unused exposure port on the exposure chamber through a Marple Model 298 Personal Cascade Impactor (Andersen Instruments, Smyrna, Georgia, USA) with a bubbler as the final stage, using a wet type gas meter. Two samples were collected during the exposure. The stainless steel collection substrates from stages 1 to 8 and the terminal bubbler stage were chemically analysed only.

Chemical Analysis Procedure
Analytical Method for the analysis of TM 09-217 in solvent trap samples:
SAMPLE ASSAY:
1. Test sample (Solvent trap collected in trapping solvent): Transfer to volumetric flask using trapping solvent
2. Further dilute, if necessary, using diluent and internal standard to provide a solution containing TM 09-217 at a nominal concentration in the range 2.0 – 100 µg/mL.
3. Inject onto the GC singly.

SAMPLE ASSAY:
1. Test sample (stainless steel plate).
2. Extract (ultrasonic vibration, 10 mins) using diluent and internal standard to provide a solution containing TM 09-217 at a nominal concentration in the range 2.0 - 100 µg/mL.
3. Inject onto the GC singly.

CALIBRATION
1. Dissolve TM 09-217 analytical reference standard (50 mg, active) in diluent (50 mL) to provide primary standard (1000 µg/ml).
2. Dilute the primary standard using diluent and Internal standard stock solution to provide calibration standards at nominal concentrations of 2, 4, 10, 20, 40, 60, 80 and 100 µg/mL.
3. Inject the eight standards onto the GC for calibration.

GC Analytical Parameters:

Diluent: Acetone.
Trapping solvent: Acetonitrile/water/antifoam, 50/50/0.02 v/v/v.
GC Conditions:
Analytical column: ZB-5MS, 10 m × 0.10 mm id., film thickness 0.10 μm
Injector temperature: 300 °C
Injector mode: Split
Split ratio: 10
Injection volume: 1 μL
Column temperature: Initial: 60°C for 1 min; Rate: 10°C/min to 180°C for 2 min
Carrier gas: Helium, 0.31 mL/min
Detector: Flame ionisation
Detector temperature: 300 ºC
Detector gas: Hydrogen, 40 mL/min; Air, 400 mL/min; Helium (make-up gas), 30 mL/min
Run time: 15 minutes
Approximate retention time: 8.9 minutes (TM 09-217); 8.8 minutes (Internal Standard)

The GC system was calibrated using external standards. Peak area ratio data acquired by the data capture software using a 1st order fit was subjected to least squares regression analysis.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Remarks on duration:

single exposure

Concentrations:

Target aerosol concentration = 5 mg/L
Mean achieved aerosol concentration = 5.25 mg/L

No. of animals per sex per dose:

3/sex/dose

Control animals:

no

Details on study design:

Serial Observations
Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in this experimental procedure were recorded in the Study Day Book. In addition, observations relating to individual animals made throughout the day were recorded.

Clinical Observations
The animals were observed intermittently for signs of reaction to the test substance during exposure and at least twice daily throughout the observation period. Clinical signs were recorded immediately prior to the start of the exposure, at approximately every hour during the remainder of exposure, immediately following exposure and then at 1 hour and 2 hours post-exposure. During the observation period, the animals were observed once in the morning and once
toward the end of the experimental day. On the day of termination observations were recorded in the morning only.

Mortality
Throughout the study, all cages were checked at least twice daily, once in the morning and again towards the end of the normal working day, for dead or moribund animals.

Body Weight
The weight of each animal was recorded once during the acclimatisation period and on Days 1 (prior to dosing), 2, 4, 8 and 15.

Necrospy
Method of Kill
Animals were killed by an overdose of penobarbitione sodium followed by exsanguination.

Macroscopic Pathology
All animals were subjected to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. Any macroscopic abnormalities in the appearance of the organs were recorded. Tissues were discarded following necropsy.

Statistics:

Computer Systems
The computer systems that were used on this study to acquire and quantify data include:

Sample Registry System*: Used for sample analysis tracking
Waters Empower 2*: Used for inhalation Analysis
Xybion Pristima*; Used for Pharmacy tet substance management
*All version numbers of the systems are maintained by Huntingdon Life Sciences

Data Treatment
In order to minimise the cumulative errors, which result from repeated rounding of numbers, some of the data in this report have been calculated continuously using unrounded numbers and only rounded for reporting. Consequently, any further calculation using these rounded numbers may include rounding errors in the last significant figure, possibly leading to small apparent discrepancies with other data in this report.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Mean aerosol concentration

Mortality:

There were no unscheduled deaths.

Clinical signs:

other: Slow breathing and closed eyelids were noted for all animals during exposure. One female also struggled during dosing. These signs were no longer noted immediately after exposure. Immediately after exposure, all animals displayed chin rubbing and one fem

Body weight:

Body weight loss was observed in all males and females on the day following the 4 hour exposure. This loss of body weight was attributed to the removal of food and water for the duration of the exposure, and was therefore not considered to be test article related.

All animals recovered from the above mentioned body weight loss by the next appropriate weighing occasion, and group mean body weights for both sexes increased from Day 4 of the observation period onwards.

Gross pathology:

The macroscopic examination performed after a single administration and a 14 day observation period revealed the following change in the lungs.

Lungs
Pale areas were seen in one female animal exposed to 5.25 mg/L. However, this macroscopic finding was not attributed to treatment, because the nature and incidence of this finding was consistent with the commonly seen background of macroscopic changes in Crl:CD (SD) rats at these laboratories.

No other abnormalities were noted.

Other findings:

Crl:CD (SD) rats were exposed to TM 09-217 for a four hour single exposure by snout only inhalation. The mean achieved aerosol concentration was 5.25 mg/L, which was 105% of target.

All treatment-related clinical signs had recovered 1 hour after exposure, there were no mortalities and no test article related effects on body weight during the exposure or the 14 day observation period. The nature and incidence of the findings noted at necropsy were consistent with the commonly seen background of macroscopic changes.

Any other information on results incl. tables

Chamber Atmosphere Conditions

Summary data are presented below:

Group	Target aerosol Con. (mg/L)	Mean achieved aerosol conc. (mg/L)	(Droplet size)   MMAD (µm)	(Droplet size) σg
1	5.0	5.25	3.6	2.16

MMAD = Mass median aerodynamic diameter

σg = Geometric standard deviation

 

The mean achieved chamber aerosol concentration was within 5% of the target concentration. The mass median aerodynamic diameter (MMAD) was within the acceptable range of 1 to 4 μm.

 

Summary of findings in the lungs for animals killed after a single administration and a 14 day observation period 

Group/sex Aerosol concentration (mg/L)	1M 5.25	1F 5.25
Pale areas	0	1
Number of animals examined	3	3

No other abnormalities were noted

Clinical Data Results

Clinical signs - group distribution of observations

Group: 1

Compound: TM 09-217

Aerosol concentration (mg/L): 5.25

Group	Signs	During Exposure	Number showing signs Time in hours
			IAE	1 hr after exposure	2 hours after exposure	Days 2 to 15
1M	Slow breathing	3	0	0	0	0
	Closed eye lids	3	0	0	0	0
	Wet fur	1	1	1	0	0
	Chin rubbing	0	3	0	0	0
1F	Struggling during dosing	1	0	0	0	0
	Slow breathing	3	0	0	0	0
	Closed eyelids	3	0	0	0	0
	Chin rubbing	0	3	0	0	0
	Wet fur	0	1	1	0	0
	Partially closed eyelidss	0	1	0	0	0

IAE = immediately after exposure

Clinical signs - individual observations

Group: 1

Compound: TM 09-217

Aerosol concentration (mg/L): 5.25  

Group	Animal No.	During Exposure	IAE	1 hour after exposure	2 hours after exposure	Days 2 to 15
1M	A1	SL, CLB	CR	Y	Y	Y
	A2	SL, CLB	CR	Y	Y	Y
	A3	SL, CLB, WF	CR, WF	WF	Y	Y
1F	A4	STR, SL, CLB	CR	Y	Y	Y
	A5	SL, CLB	CR, WF, PCB	WF	Y	Y
	A6	SL, CLB	CR	Y	Y	Y

IAE = Immediately after exposure

SL = Slow breathing

CLB = Closed eyelids

CR = Chin rubbing

WF = Wet fur

STR = Struggling during dosing

PCB = Partially closed eyelids

Y = Within normal limits

Body Weights –Individual and group mean values (g)

Group: 1

Compound: TM 09-217

Aerosol concentration (mg/L): 5.25

Group	Animal No.	-7	1	2	4	8	15
1M	A1	471	502	495	503	520	549
	A2	430	450	439	452	468	494
	A3	470	507	492	498	506	532
	Mean	457	486	475	484	498	525
	SD	23.4	31.6	31.5	28.1	26.9	28.2
1F	A4	270	278	268	272	279	297
	A5	278	286	276	278	279	287
	A6	247	260	246	260	263	269
	Mean	265	275	263	270	274	284
	SD	16.1	13.3	15.5	9.2	9.2	14.2

Macroscopic pathology – individual findings

Group: 1

Compound: TM 09-217

Aerosol concentration (mg/L): 5.25

 

Group	Animal No.	Region/organ affected	Observation
1F	A6	Lungs	Pale areas, multiple, up to 1 mm, sub pleural

 

Aerosol Technology Report Results

Chamber Atmosphere Conditions

Achieved chamber TM 09-217 aerosol concentrations are presented below:

Sample Number	Actual sample time point (minutes)	Achieved chamber aerosol concentration (mg/L)
1	17	4.63
2	54	5.12
3	124	5.58
4	180	5.43
5	214	5.51
Mean	-	5.25
SD	-	0.391

SD Standard deviation

The mean achieved aerosol concentration value was close to the target of 5 mg/L.

Droplet Size Distribution

Calculated Mass Median Aerodynamic Diameter and Geometric Standard Deviation values for aerosolized TM 09-217 are presented in the following tables:

Average achieved chamber aerosol conc. (mg/L)	MMAD (µm)	GSD	%<7 µm
5.25	3.6	2.16	80

MMAD Mass median aerodynamic diameter

GSD Geometric standard deviation

The mean MMAD value was within the ideal range of 1 to 4 microns.

Stage No.	ECD (µm)	Percentage of TM 09-217 collected on each stage
		PSD number          1                        2                      Mean
1	21.3	0.8	1.5	1.2
2	14.8	1.0	1.2	1.1
3	9.80	5.9	8.7	7.3
4	6.00	14.8	18.9	16.9
5	3.50	27.6	34.8	31.2
6	1.55	34.5	27.3	30.9
7	0.93	13.1	5.8	9.4
8	0.52	1.3	0.0*	0.6
Filter	0.00	1.1	1.9	1.5
MMAD		3.4	3.9	3.6
σg		2.10	2.19	2.16

ECD Effective cut off diameter of cascade impactor stage at 2 L/min

PSD Particle size distribution

MMAD Mass median aerodynamic diameter (μm)

GSD Geometric standard deviation

* No peak detected. Result reported as zero

Chamber Air Temperature

 

Chamber air temperature was measured using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. The chamber air temperatures during exposure are presented below:

 

Time (Minutes)	Chamber Air Temp (°C)
0	21.9
30	22.2
60	22.5
90	22.6
120	22.4
150	22.5
180	22.5
210	22.3
240	22.4
Mean	22.4
SD	0.21

SD = standard deviation

 

Chamber air temperature values were within the guideline recommended range.

Chamber Relative Humidity

Chamber relative humidity was measured using an electronic hygrometer probe inserted into the breathing zone of the animals via an unused exposure port. The chamber relative humidity values during exposure are detailed below: 

Time (Minutes)	Chamber relative humidity (%)
0	45.8
30	46.8
60	47.4
90	46.4
120	38.8
150	45.4
180	47.2
210	46.5
240	45.9
Mean	45.9
SD	2.62

SD = standard deviation

 

Relative humidity values were within the guideline recommended range.

Nominal Concentration

The nominal concentrations are presented in the following table.

System Airflow (L/minute)	Air Changes (per hour)*	Generation Period (mins)	Usage (g)	Nominal Con. (mg/L)**
20	40	245	71.46	14.6

* Airflow sufficient to given more than 12 air changes over hour with air supply containing >19% oxygen

Air changes per hour = (60 (mins) x Airflow (L/min)) / (Chamber Volume (L))

Chamber volume = 30 L

** Nominal concentration (mg/L) = (Usage (g) x 1000) / (Generation period (mins) x Airflow (L/min))

The T₉₅equilibration time was 5 minutes

Time (mins) to reach T₉₅= Natural Logarithm (100 / (100-95)) x (Chamber Volume (L) / (Airflow (L/min))

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Crl:CD (SD) rats were exposed to TM 09-217 for a four hour single exposure by snout only inhalation. The mean achieved aerosol concentration was 5.25 mg/L, which was 105% of target.

There were no mortalities during this study, therefore according to the Globally Harmonized Classification System (GHS; UNITED NATIONS) TM 09-217 is unclassified.

Executive summary:

The acute inhalation toxicity of the test substance TM 09-217 was assessed and had an LC50 of > 5.25 g/L according to the OECD Test Guideline 436 using the acute toxic class method.