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

Endpoint summary

Description of key information

The acute oral toxicity of the test substance, TM 09-217, was assessed and gave an LD50 of > 2000 mg/kg body weight according to the OECD Test Guideline 423 using an acute toxic class method. 
The acute dermal toxicity potential of the test substance, TM 09-217 was assessed and gave an LD50 of > 2000 mg/kg body weight according to OECD Test Guideline 402, using a fixed dose method. 
The acute inhalation toxicity potential of the test substance, TM 09-217 was assessed and gave an LC50 of > 5.25 mg/L according to OECD Test Guideline 436 using the acute toxic class method. 

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:

The study was conducted between 25 February 2014 and 27 March 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 Guideline 423 using the acute toxic class method and in compliance with GLP.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

female

Details on test animals or test system and environmental conditions:

Healthy nulliparous and non-pregnant female Crl:CD (SD) albino rats were obtained from a reputable supplier The animals were allocated without conscious bias to cages within the treatment groups and were housed in groups of three. Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark. The animals were allowed to acclimatise to the conditions described below for at least 5 days before treatment. For those animals selected for this study, their body weights were in the range 205 to 234 g and they were approximately eight to twelve weeks of age prior to dosing (Day 1).

Animal Housing, diet and water supply
Animals were housed inside a barriered 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. The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23 °C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.
The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fibre bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.

The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except for overnight prior to and approximately four hours after dosing. 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 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 diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Formulation
The test substance was formulated at a concentration of 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg body weight.
The test substance formulations were prepared on the day of dosing. The absorption of the test substance was not determined.
Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study.Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Administration
The appropriate dose volume of the test substance was administered to each rat by oral gavage using a plastic syringe and plastic catheter.
A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. Formulations were stirred before and throughout the dosing procedure.

Doses:

300 mg/kg and 2000 mg/kg

No. of animals per sex per dose:

Three females per group.

Control animals:

no

Details on study design:

Mortality
Cages of rats were checked at least twice daily for any mortalities.

Clinical Observations
Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, surviving animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation. All surviving animals were observed for 14 days after dosing.

Body Weight
The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15 or at death. Individual weekly body weight changes and group mean body weights were calculated.

Necropsy
Method of kill
All surviving animals were humanely killed on Day 15 by carbon dioxide asphyxiation.

Macroscopic pathology
All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.

Statistics:

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

System name*: Xybion Pristima
System function: Used for Pharmacy test substance management

* All version numbers of the systems are maintained by Huntingdon Life Sciences

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

One female (An. No. C2) dosed at 300 mg/kg died on Day 5. Clinical signs prior to death comprised piloerection, unsteady gait, fast breathing, irregular breathing, hunched posture, pallor of the extremities, extremities blue in colour and lachrymation. These signs were seen from the second day after dosing. A loss in body weight was noted for the decedent between dosing and death. Macroscopic examination of the animal revealed slightly pale contents of the thoracic cavity along with gelatinous opaque material surrounding the lungs and heart and a small amount of pale serous fluid present. Oesophagus had a small perforation at the level of the diaphragm and gaseous distension in the GI tract.
This death was not considered to be an effect of the test material and was due to an intubation error during the dosing procedure.
There were no other deaths throughout the study.

Clinical signs:

other: Clinical signs of reaction to treatment in females dosed at 2000 mg/kg comprised piloerection (all animals), under activity (An. Nos. C8 – C12), hunched posture ( An. Nos. C8, C10 – C12), irregular breathing and elevated gait (An. No C8) and loose faeces

Gross pathology:

Pallor of the kidneys was noted in two females (An. Nos. C4 and C5) dosed at 300 mg/kg at study termination. No other abnormalities were noted in any of the surviving animals at the macroscopic examination at study termination on Day 15.

Mortality data

Dose (mg/kg)	No. of deaths in groups of 3	1 to 4 a                 b	Day 5*  a                   b	6 to 15      a                 b
300	1/ 3 F	0                  0	1                    0	0                 0
300	o/3 F	-                   -	-                     -	-                  -
2000	0/3 F	-                   -	-                     -	-                  -
2000	0/3 F	-                   -	-                     -	-                  -

 

*The day/time indicated is the time that the animal was found dead

F = Female

a = First observation

b = Second observation

- = Not applicable

Clinical Signs

Clinical Signs	No. of rats in groups of 3 showing signs Dose (mg/kg)
		300		3000
Piloerection		1	0	3	3
Underactive behaviour		0	0	2	3
Hunched posture		1	0	1	3
Loose faeces		0	0	1	2
Irregular breathing		1	0	1	0
Elevated gait		0	0	1	0
Unsteady gait		1	0	0	0
Fast breathing		1	0	0	0
Extremities blue in colour		1	0	0	0
Pallor of the skin		1	0	0	0
Lachrymation		1	0	0	0

Individual and group mean body weights (g)

Dose (mg/kg)	Sex	Animal Number	Body weights (g) at Day
			1*	8	15	Death
300	Female	C1	215	248	260	-
		C2	234	-	-	199
		C3	217	245	264	-
		Mean	222	247	262	-
	Female	C4	210	241	249	-
		C5	228	250	264	-
		C6	211	237	246	-
		Mean	216	243	253	-
2000	Female	C7	217	255	264	-
		C8	205	242	247	-
		C9	207	235	245	-
		Mean	210	244	252	-
	Female	C10	219	257	268	-
		C11	215	250	274	-
		C12	212	234	246	-
		Mean	215	247	263	-

* Prior to dosing

-  Not applicable

Individual body weight changes (g)

Dose (mg/kg)	Sex	Animal Number	Body weight changes (g) at Days
			1-8	8-15
300	Female	C1	33	12
		C2	-	-
		C3	28	19
	Female	C4	31	8
		C5	22	14
		C6	26	9
2000	Female	C7	38	9
		C8	37	5
		C9	28	10
	Female	C10	38	11
		C11	35	24
		C12	22	12
-              Not applicable

 

Macroscopic Findings

Dose (mg/kg)

300

2000

Female

Female

Animal Number

C1

C2

C3

C4

C5

C6

C7

C8

C9

C10

C11

C12

Fate of animal

K

D

K

K

K

K

K

K

K

K

K

K

Day of death

15

5

15

15

15

15

15

15

15

15

15

15

Tissues examined

Findings

Subcutaneous tissue

N

2

N

N

N

N

N

N

N

N

N

N

Brain

N

N

N

N

N

N

N

N

N

N

N

N

Heart

N

9, 10

N

N

N

N

N

N

N

N

N

N

Lungs

N

9, 10

N

N

N

N

N

N

N

N

N

N

Liver

N

N

N

N

N

N

N

N

N

N

N

N

Spleen

N

N

N

N

N

N

N

N

N

N

N

N

Kidneys

N

N

N

4

4

N

N

N

N

N

N

N

Stomach

N

11

N

N

N

N

N

N

N

N

N

N

Duodenum

N

8

N

N

N

N

N

N

N

N

N

N

Small Intestines

N

8

N

N

N

N

N

N

N

N

N

N

Large Intestines

N

8

N

N

N

N

N

N

N

N

N

N

Caecum

N

8

N

N

N

N

N

N

N

N

N

N

Urinary Bladder

N

N

N

N

N

N

N

N

N

N

N

N

K            Killed at study termination D            Sporadic death N            No abnormalities detected 2             Congestion (darkened tissue/organs) 4             Pallor of tissues/organs 8             Gaseous distension 9             Slightly pale contents of the thoracic cavity and a small amount of pale serous fluid present 10          Gelatinous opaque material surrounding the lungs and heart 11          Small perforation of the oesophagus at the level of the diaphragm 12          Gaseous distension in the gastro intestinal tract

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute median lethal oral dose (LD50) to rats of TM 09-0217 was demonstrated to be greater than 2000 mg/kg body weight.
TM 09-0217 is included in Category 5 according to the Globally Harmonised System (GHS).

Executive summary:

The acute oral toxicity of the test substance, TM 09-217, was assessed and gave an LD50 of > 2000 mg/kg body weight according to the OECD Test Guideline 423 using an acute toxic class method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

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:

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.

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

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.

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.

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.

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))

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5.25 mg/m³ air

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 25 February 2014 and 02 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 402 using a fixed dose method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute dermal toxicity (2-1-2), 12 Nousan No 8147, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: CD (Crl:CD ‘SD’)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal supply, acclimitisation and allocation
Healthy male and female (nulliparous and non-pregnant) CD (Crl:CD ‘SD’) albino rats were obtained from a reputable supplier.
The animals were allocated without conscious bias to cages within the treatment group. They were housed individually from Day -1 until Day 13 when they were returned to group housing (in groups of five rats of the same sex). The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.

Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark.

The animals were allowed to acclimatise to the conditions described below for 6 days before treatment. For those animals selected for this study, their body weights were in the range 347 to 388 g for males and 246 to 271 g for females and they were approximately eight to twelve weeks of age prior to dosing (Day 1).

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 044/045). The facility was designed and operated to minimise the entry ofexternal biological and chemical agents and to minimise the transference of such agents between rooms.

The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23 °C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.

The 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 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 diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

A group of ten rats (five males and five females) was treated at 2000 mg/kg body weight.

One day prior to treatment, hair was removed from the dorso-lumbar region of each rat with electric clippers taking care to avoid damaging the skin, exposing an area equivalent to approximately 10% of the total body surface area.

The test substance was applied by spreading it evenly over the prepared skin. The treatment area (approximately 50 mm x 50 mm) was covered with porous gauze held in place with a non-irritating dressing, and further covered by a waterproof dressing encircled firmly around the trunk of the animal.

Treatment in this manner was performed on Day 1 (day of dosing) of the study only.

At the end of the 24 hours exposure period the dressing was carefully removed and the treated area of skin was washed with warm water (30 - 40°C), to remove any residual test substance. The treated area was blotted dry with absorbent paper.

A record of the weight of the test substance dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.

TM 09-0217 was administered, as supplied, at a dose volume of 2000 mL/kg body weight. The specific gravity, as measured by this laboratory, was 0.872 g/mL. The absorption of the test substance was not determined. Determination of the homogeneity, stability and purity of the test substance was not undertaken as part of this study.

Duration of exposure:

24 hours

Doses:

Single dose 2000 mg/kg

No. of animals per sex per dose:

5 female and 5 male per dose

Control animals:

no

Details on study design:

Serial Observations
Mortality:
Cages of rats were checked at least twice daily for any mortalities.

Clinical Observations:
Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation. All animals were observed for 14 days after dosing.

Bodyweight:
The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15. Individual weekly body weight changes and group mean body weights were calculated.

Necropsy and macropathology
Method of kill:
All animals were humanely killed on Day 15 by carbon dioxide asphyxiation

Macroscopic pathology:
All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.

Statistics:

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

System name: Xybion Pristima*
System Function: Used for Pharmacy test substance management

* All version numbers of the system are maintained by Huntingdon Life Sciences

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths and no systemic response to treatment in any animal.

Clinical signs:

other: Very slight to well-defined erythema was seen in one male (An. No. A4) and four females (An. Nos A7 to A10). These reactions had resolved by Day 9. In addition, eschar/scab formation was seen in one female (An. No. A9) from Day 7, resolving completely b

Gross pathology:

No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute median lethal dermal dose (LD50) to rats of TM 09-0217 was demonstrated to be greater than 2000 mg/kg body weight.

Executive summary:

The acute dermal toxicity potential of the test material, TM 09-217 was assessed and gave an LD₅₀ of > 2000 mg/kg body weight according to OECD Test Guideline 402, using a fixed dose method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Acute oral toxicity:

One female dosed at 300 mg/kg died on Day 5. This death was considered to be procedural in origin (as a perforated oesophagus was evident at the macroscopic examination) and not an effect of the test substance. There were no other deaths throughout the study. Clinical signs of reaction to treatment in females dosed at 2000 mg/kg comprised piloerection under activity, hunched posture, irregular breathing and elevated gait and loose faeces. These signs were first noted approximately one hour after dosing. Recovery of surviving animals, as judged by external appearance and behaviour, was complete by Day 3. No clinical signs were seen in any of the surviving animals dosed at 300 mg/kg. All surviving animals were considered to have achieved satisfactory body weight gains throughout the study. Pallor of the kidneys was noted in two females dosed at 300 mg/kg at study termination. No other abnormalities were noted in any of the surviving animals at the macroscopic examination at study termination on Day 15.

Acute inhalation toxicity:

There were no unscheduled deaths. 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 female had partially closed eyelids. These signs were no longer evident 1 hour after exposure. 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. Group mean body weights for both sexes increased from Day 4 of the observation period onwards. The macroscopic examination performed after a single administration and a 14 day observation period revealed no treatment-related abnormalities. Pale areas were observed in the lungs of one female exposed to 5.25 mg/L; however the nature and incidence of this finding was consistent with the commonly seen background of macroscopic changes.

Acute dermal toxicity:

There were no deaths and no systemic response to treatment in any animal. Very slight to well-defined erythema was seen in one male and four females. These reactions had resolved by Day 9. In addition, eschar/scab formation was seen in one female from Day 7, resolving completely by Day 13. A bodyweight loss was noted for one female on Day 8 and a low body weight gain was noted for one female on Day 8 and two females on Day 15. All other animals were considered to have achieved satisfactory body weight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Justification for selection of acute toxicity – oral endpoint
The study was conducted in vivo in an appropriate test species according to internationally recognised guidelines.

Justification for selection of acute toxicity – inhalation endpoint
The study was conducted in vivo in an appropriate test species according to internationally recognised guidelines.

Justification for selection of acute toxicity – dermal endpoint
The study was conducted in vivo in an appropriate test species according to internationally recognised guidelines.

Justification for classification or non-classification

Substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. Acute toxicity values are expressed as approximate LD50 or LC50 (inhalation) values. 

A test substance is classified according to one of these four toxicity categories when the acute LD50 value is ≤ 2000 mg/kg for exposure via the oral and dermal routes.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute oral LD50 of > 2000 mg/kg and therefore the test substance, TM 09-217, is not classified for acute oral toxicity.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute dermal LD50 of > 2000 mg/kg and therefore the test substance, TM 09-217, is not classified for acute dermal toxicity.

An in vivo study performed according to internationally recognised guidelines and conducted according to GLP gave an acute inhalation LC50 of > 5.25 mg/mL and therefore the test substance, TM 09-217, is not classified for acute inhalation toxicity.