Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 15 May 2014 and 14 August 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 403 using a standard acute 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:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identification: IFF TM 12-209
Physical state/appearance: Extremely pale yellow liquid
Storage conditions: Stored cold at approximately 4 °C, in the dark

Test animals

Species:
rat
Strain:
other: RccHanTM : WIST
Sex:
male/female
Details on test animals and environmental conditions:
Male and female RccHan™ : WIST strain rats were supplied by Harlan Laboratories UK Ltd, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately eight to twelve weeks old and within the weight range of 200g to 350g. The females were nulliparous and non-pregnant.

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

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

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

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

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Atmosphere Generation
The test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, 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 controlled by adjusting the rate of the infusion pump. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.

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

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied to achieve the required atmospheric conditions.


Exposure Procedure
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 the test item for a period of four hours. A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 108% of target and no deaths occurred, no further levels were required.


Sighting Exposure
During characterization, a group of two rats (one male, one female) were exposed to an atmosphere of the test item at a mean achieved atmosphere concentration of 2.71 mg/L for approximately four hours. Increased respiratory rate was the only significant observation noted in both animals on removal from the chamber and two hours after the four hour exposure period had been completed. One day post-exposure both animals exhibited increased respiratory rate, hunched posture and pilo-erection. Both animals recovered to appear normal on either Day 4 or Day 5 post-exposure.

Exposure Chamber Atmosphere Concentration
Prior to the start 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 between 19 and 21 C 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 non-volatile component of the batch used during the study was found to be approximately 0.49% (n=10). Due to the very low proportions of non-volatiles in the test item it was considered that chemical analysis should be employed in order to determine test atmosphere concentrations.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved two liters of test atmosphere being drawn through a glass impinger containing Methanol (each made up to 80 mL). The samples were then submitted for chemical analysis.

The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.

The nominal concentration was 223 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straightforward.

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 (9.6, 6.9, 4.2, 1.7, 0.93 and 0.43 µm cut points) with stainless steel collection substrates and a back up glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.

The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
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 9.6, 6.9, 4.2, 1.7, 0.93 and 0.43 µm was calculated.

The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.42 mg/L
No. of animals per sex per dose:
Five / sex / dose
Control animals:
no
Details on study design:
Clinical Signs
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of overt toxicity was recorded at each observation.

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

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

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.42 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no deaths during the study.
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations a
Body weight:
All animals exhibited body weight losses or showed no body weight gains on the first day post-exposure. Reasonable body weight gains were noted in all male animals during the remainder of the recovery period. In contrast, two females exhibited a slight body weight loss or no body weight gain from Days 1 to 3 post-exposure. Three females either showed a slight body weight loss or showed no body weight gain from Days 3 to 7 post-exposure, two females exhibited slight body weight losses during the final week of the recovery period.
Gross pathology:
No macroscopic abnormalities were detected amongst animals at necropsy.

Any other information on results incl. tables

Particle Size Distribution

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

 

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

5.42

3.89

51.4

2.22

 

Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by high performance gas chromatography (GC). The test atmosphereswere sampled after theoretical chamber equilibration and then at approximately half-hourly intervals during the exposure period.

 

The concentration of the test item was shown to be stable and the mean values obtained were:

 

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

5.42

0.09

12.1

 

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

 

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

[1]= Test atmospheres were generated for a total of 12 minutes prior to animal insertion to ensure test item

Exposure Chamber Atmosphere Concentrations

Duration of Exposure (minutes)

Volume of Air Sampled (L)

Chamber Flow Rate (L/min)

Atmosphere Concentration (mg/L)

5

2

60

5.27

31

2

60

5.27

60

2

60

5.48

93

2

60

5.44

120

2

60

5.54

150

2

60

5.43

179

2

60

5.44

210

2

60

5.43

235

2

60

5.46

 

Mean achieved atmosphere concentration (mg/L) =5.42

Standard deviation =0.09

 

Nominal concentration:

Test item used (g)

183

Air Flow (L/min)

60

Total Generation Time (mins)

252[1]

Nominal Concentration (mg/L)

12.1

 

Particle Size Distribution

Cascade Impactor Data

Impactor Stage Number

Cut Point

(µm)

Amount Collected (mg) per Sample Number

Mean Amount Collected (mg)

1

2

3

3

9.6

0.05

0.03

0.10

0.06

4

6.9

0.27

0.15

0.35

0.26

5

4.2

0.56

0.49

0.56

0.54

6

1.7

0.20

0.02

0.21

0.14

7

0.93

0.01

0.00

0.10

0.04

8

0.43

0.01

0.00

0.05

0.02

Back-up Filter

<0.43

0.02

0.03

0.03

0.03

Total Mean Amount of Test Item Collected

1.09

 

Calculation

Cut Point

(µm)

Log10

Cut Point

Mean Cumulative Amount Less Than Cut Point

(mg)

(%)

Probit

9.6

0.982

1.03

94.5

6.60

6.9

0.839

0.77

70.6

5.54

4.2

0.623

0.23

21.1

4.20

1.7

0.230

0.09

8.26

3.61

0.93

-0.032

0.05

4.59

3.31

0.43

-0.367

0.03

2.75

3.08

 

Results

Mean Mass Median Aerodynamic Diameter (MMAD) =3.89µm

Geometric Standard Deviation (GSD) =2.22

Predicted amount less than 4 µm =51.4%


[1]= Test atmospheres were generated for a total of 12 minutes prior to animal insertion to ensure test item concentration was being achieved.

Mortality Data

Mean Achieved Atmosphere Concentration (mg/L)

Sex

Deaths During Exposure

Deaths Post Exposure

(1 Hour)

Deaths During Day of Observation

Total Deaths

1

2

3

4

5

6

7

8-14

5.42

Male

0

0

0

0

0

0

0

0

0

0

0/10

Female

0

0

0

0

0

0

0

0

0

0

 

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No deaths occurred in a group of ten rats (five males and five females) exposed to a mean achieved atmosphere concentration of 5.42 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of IFF TM 12-209, in the RccHanTM : WIST strain rat, was greater than 5.42 mg/L.
Executive summary:

The acute inhalation toxicity of the test substance, TM 12-209, was assessed according to OECD Test Guideline 403 using a standard acute method and gave an acute inhalation median lethal concentration (4 hr LC50) of greater than 5.42 mg/L according to OECD Test Guideline 403 using a standard acute method.