Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July to 09 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 403 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 12-14 March 2014 / signed on 12 May 2014)
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
700-902-0
EC Number:
700-902-0
Cas Number:
1370699-98-1
Molecular formula:
C12H14O3
IUPAC Name:
700-902-0
Test material form:
liquid
Details on test material:
- Physical state: Colourless liquid
- Water solubility: 2.16 g/L
- Vapour pressure: 0.33 Pa (at 20°C) and 0.59 Pa (at 25°C)
- Partition coefficient: 2.69 (HPLC, 2013)
- Storage Conditions: 6 ± 2 °C, protected from light, under nitrogen in the original container
- Stability under test conditions: Not specified, assumed to be stable
Specific details on test material used for the study:
- Purity test date: 30 October 2013
- Storage Conditions: ca. 4 °C in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan™: WIST strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 8-12 weeks
- Weight at study initiation: 198-350 g
- Housing: Animals were housed in groups of up to five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes.
- Diet: Food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 per hour
- Photoperiod: 12 h dark / 12 h light

N-LIFE DATES: 17 July to 09 October 2014

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.63 µm
Geometric standard deviation (GSD):
2.57
Remark on MMAD/GSD:
Predicted amount less than 4 μm = 67.3 %
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber.
- Exposure chamber volume: Cylindrical exposure chamber had a volume of approximately 30 L (dimensions: 28 cm diameter x 50 cm high).
- One day prior to the day of exposure each rat was acclimatized (for approximately 2 h) to a tapered polycarbonate restraining tube.
- Method of holding animals in test chamber: 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.
- Source and rate of air: Chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times (33, 94 and 214 minutes of exposure respectively) during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK).
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature and humidity in air chamber: 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. Temperature and humidity in air chamber were 21 °C and 37-39%, respectively.
- 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.
- 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 20 and 22 °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 91.06 % (n=10). However, once a drying technique had been employed on filters sampled from the generated test atmosphere it was apparent that the concentration calculated was much lower than the results noted on the test filters prior to drying and as such it was concluded that the non-volatile content was being affected by the generation method. It was therefore considered that chemical analysis should be employed in order to determine test atmosphere concentrations. The test atmosphere was sampled nine times (5, 30, 60, 90, 120, 150, 180, 210 and 235 minutes of exposure respectively) during the exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a series of two glass impingers containing Hexane (each made up to 80mL).
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 598 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.
- Pretreatment: 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.
- 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 119 % of target, the standard deviation of the test atmosphere concentrations was within acceptable limits and no deaths occurred, no further levels were required.

TEST ATMOSPHERE
- Brief description of analytical method used: Test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.
- Samples taken from breathing zone: Yes
- Particle size distribution: See table 7.2.2/1
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.63 μm / 2.57
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Nominal concentration: 35.7 mg/L
Mean achieved atmosphere concentration: 5.97 mg/L
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Duration of observation period following administration: 14 days
- Frequency of observations: 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 deaths or evidence of overt toxicity were recorded at each observation.
- Frequency of weighing: Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14 or at death.
- Necropsy of survivors performed: Yes, at the end of the 14 day 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.
Statistics:
None

Results and discussion

Preliminary study:
Not applicable
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.97 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: Only one death occurred in a group of ten rats
Mortality:
Only one death occurred in a group of ten rats at 5.97 mg/L air.
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 gain on the first day post-exposure. Body weight gains were noted in all animals during the remainder of the recovery period, with the exception of one female animal (Animal No. 6) which showed no body weight gain from Days 1 to 3 post-exposure.
Gross pathology:
No macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period, with the exception of one surviving male (Animal No. 4) which exhibited dark patches on the lungs.
The following macroscopic abnormalities were detected in the animal (Animal No. 8) that was humanely killed during the course of the study at necropsy:
Liver – pale;
Kidneys – pale.
Due to the observations noted it is considered that the death noted during the study may have been mainly attributable to systemic toxicity.
Other findings:
None

Any other information on results incl. tables

Exposure Chamber Concentration:

The actual concentration of the test item was measured off-line by high performance gas chromatography (GC). The test atmospheres were 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)

 

5.97

0.25

35.7

 

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).

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

 

5.97

2.63

67.3

2.57

 

Table 7.2.2/1: 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

5.51

3

2

60

5.86

60

2

60

5.82

90

2

60

6.11

120

2

60

6.36

150

2

60

6.03

180

2

60

6.21

210

2

60

5.93

235

2

60

5.86

Mean achieved atmosphere concentration (mg/L) = 5.97

Standard deviation = 0.25

Nominal concentration:

Test item used (g)

535

Air Flow (L/min)

60

Total Generation Time (mins)

250*

Nominal Concentration (mg/L)

35.7

  * = Test atmospheres were generated for a total of 10 minutes prior to animal insertion to ensure test item concentration was being achieved.

Table 7.2.2/2: 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

5.97

Male

 

0

0

0

0

0

0

0

0

0

0

1/10

Female

0

0

1*

0

0

0

0

0

0

0

* = Humanely killed as considered unlikely to survive

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
Under the test conditions, the substance is:
- not classified according to the Regulation EC No. 1272/2008 (CLP) as the LC50 is higher than 5.97 mg/L.
- classified as 'category 5' according to the GHS based on significant clinical signs observed at 5.97 mg/L.
Executive summary:

In an acute inhalation toxicity study performed according to OECD Guideline 403, groups (5/sex/dose) of RccHan: WIST strain rats were exposed (nose only) to aerosol atmosphere of ST 14 C 12 at concentration of 5.97 mg/L (mean achieved) for 4 h. Animals were observed for mortality, clinical signs and body weight for 14 days and at the end of the study the animals were sacrificed for macroscopic examination.

 

Observation changes noted during the study in the surviving animals included decreased respiratory rate, hunched posture, pilo-erection and wet fur. Frequent instances of increased respiratory rate and lethargy, occasional instances of labored respiration, noisy respiration, comatose and dehydration and isolated instances of ataxia, dry eyes (blinking less than usual with no blinking on contact with the eyes) and pallor of the extremities were also noted. Surviving animals recovered to appear normal on Day 8 post-exposure. Observations noted in the animal that was humanely killed during the study included decreased respiratory rate, labored respiration, noisy respiration, comatose, dehydration, pilo-erection and wet fur.

 

All animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. Body weight gains were noted in all animals during the remainder of the recovery period, with the exception of one female animal which showed no body weight gain from Days 1 to 3 post-exposure.

No macroscopic changes were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period, with the exception of one surviving male animal which exhibited dark patches on the lungs. The following macroscopic changes were detected in the animal that was humanely killed during the course of the study at necropsy: Liver and kidneys – pale. Due to the observations noted it is considered that the death noted during the study may have been mainly attributable to systemic toxicity.

 

Only one death occurred in a group of ten rats (five males and five females) exposed to a mean achieved atmosphere concentration of 5.97 mg/L for four hours. Therefore, the acute inhalation median lethal concentration (4 h LC50) of ST 14 C 12 was greater than 5.97 mg/L.

LC50(combined-rats) > 5.97 mg/L

Under the test conditions, the substance is:

- not classified according to the Regulation EC No. 1272/2008 (CLP) as the LC50 is higher than 5.97 mg/L.

- classified as 'category 5' according to the GHS based on significant clinical signs observed at 5.97 mg/L.

This study is considered as acceptable and satisfies the requirement for acute inhalation toxicity endpoint.