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Description of key information

Acute oral toxicity: The test substance was found to have a LD50 greater than 2000 mg/kg bodyweight.
Acute inhalation toxicity: The test substance was found to have an LC50 greater than 5.92 mg/L.

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:
27 March 2012 and 19 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Qualifier:
according to
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, Uk
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: the bodyweight variation did not exceed ± 20 % of the bodyweight if the initially dosed animal
- Fasting period before study: over night before dosing and for approximately 3 to 4 hr after dosing
- Housing: The animals were housed in groups of up to 4 in suspended solid-floor polypropylene cages furnished with woodflakes. The animals were provided with environmental enrichment items.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Doses:
2000 mg/kg
No. of animals per sex per dose:
5/sex/dose
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: ½, 1, 2 and 4 hr after dosing and subsequently once daily for 14 d. Morbidity and mortality checks were made daily.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths
Clinical signs:
Decreased respiratory rate was noted 1 and 2 d after dosing in the initial treated animal. There were no signs of systemic toxicity noted in the remaining animals.
Body weight:
All animals showed expected gains in bodyweight over the observation period.
Gross pathology:
A cavity in the right kidney, which was considered to be a genetic defect and not test item related, was noted at necropsy of 1 animal. No abnormalities were noted at necropsy of the remaining animals.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute toxicity of the test item was assessed via the oral route according to OECD guideline 420. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
Three separate acute toxicity studies via the oral route have been conducted on the test material, namely Harlan Laboratorties Ltd (2012), Sauer & Robbins (1979) and Wohl (1974). The Harlan Laboratories Ltd (2012) study was conducted according to OECD guideline 420 and GLP and is well documented and has been assigned reliability 1. The Sauer & Robbins (1979) study meets generally accepted scientific standards and is well documented and has been assigned reliability 2. The Wohl (1974) study does not provide sufficient information for assessment and has been assigned reliability 4.

The results of the Sauer & Robbins (1979) and Wohl (1974) studies support the result that the test item is not considered to be acutely toxic via the oral route.

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 performed between 19 September 2012 and 08 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
other: RccHan™: WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd UK Ltd, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 200 g - 360* g (* = Due to availability, one male animal was outside the weight range specified in the study plan (200- 350g). This deviation was considered not to affect the purpose or validity of the study)
- Housing: groups of 3 by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 25
- Humidity (%): 30- 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical exposure chamber. 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
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Prior to the day of exposure each rat was acclimatised (for approximately 2 h) to a tapered polycarbonate restraining tube. During the day of exposure, 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.
- Source and rate of air: 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 nebuliser.
- System of generating particulates/aerosols: The test item was aerosolised using a glass concentric jet nebuliser located at the top of the exposure chamber. The nebuliser was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Temperature, humidity: 20 °C, 72-74 %
- Oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser located in a port in the animals breathing zone during the 4-h exposure period. The test atmosphere was generated to contain at least 19% oxygen

TEST ATMOSPHERE
- Brief description of analytical method used: GC analyis
- Samples taken from breathing zone: yes; the test atmosphere was sampled 5 times during the exposure period

TEST ATMOSPHERE
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined 3 times during the exposure period using a Marple Personal Cascade. This device consisted of 6 impactor stages (8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 μm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 8.6, 5.5, 3.8, 1.7, 0.86 and 0.41 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.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.78 µm/ 3.02
- Predicted amount less than 4 µm: 76.9 %

CLASS METHOD
- Rationale for the selection of the starting concentration: A target concentration of 5.0 mg/L was used for the exposure. As the mean achieved concentration was 118% of target and no deaths occurred, no further levels were required.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
A target concentration of 5.0 mg/L was used for the exposure.
No. of animals per sex per dose:
3/ sex/ dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: clinical signs, all animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 h after termination of exposure and subsequently once daily for 14 d. Any evidence of overt toxicity was recorded at each observation; body weight,individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1,3, 7 and 14
- Necropsy of survivors performed: yes, at the end of the 14 d 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
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.92 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no mortalities.
Clinical signs:
One day after exposure, all animals exhibited increased respiratory rate, hunched posture and pilo-erection. Animals recovered to appear normal from Days 5 to 7 postexposure.
Body weight:
All male animals and 1 female exhibited slight bodyweight losses or showed no bodyweight gain on the first day post-exposure. Bodyweight gains were noted in all animals during the remainder of the recovery period, with the exception of 1 female animal which exhibited a bodyweight loss from Days 1 to 3 and showed no bodyweight gain from Days 3 to 7 post-exposure.
Gross pathology:
No macroscopic abnormalities were detected amoungst animals at necropsy.

Exposure Chamber Concentration

The actual concentration of the test item was measured off-line by Gas Chromatography (GC). The test atmospheres were sampled after theoretical chamber equilibration and then at approximately hourly intervals during the exposure period. The mean values obtained were:

Atmosphere concentration

Mean achieved (mg/L)

Standard deviation

Nominal (mg/L)

5.92

0.64

8.39

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour. The theoretical chamber equilibration time (T99) was 3 min* (* = The test atmosphere was generated for a total of 30 minutes prior to animal insertion to ensure test item concentration was being achieved.)

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 (5 <4 µm)

Geometric standard deviation

5.92

1.78

76.9

3.02

The GSD is out of the target range that would generally be acceptable for this type of study (1.5 – 3.0). This deviation was very slight (+ 0.02) and is considered to be due to the physical characteristics of the test item. However, as the particle size distributions are far lower than 4μm this is extremely small deviation to the test guideline is considered not to affect the purpose or validity of this study.

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute toxicity of the test item was assessed via the inhalation route according to OECD guideline 436. No deaths occured in a group of 6 rats exposed to the mean acheived concentration of 5.92 mg/L for 4 h. Therefore, the acute inhalation median lethal concentration (4 h LC50) of the test item in the RCCHan™:WIST strain rat was greater than 5.92 mg/L.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Acute toxicity via the oral route: An acute oral toxicity study was performed in the rat in accordance with GLP and OECD Guideline 420. Following a sighting test at a dose level of 2000 mg/kg, 4 fasted female Wistar strain rats were given a single oral dose of test item at a dose level of 2000 mg/kg bodyweight. There were no mortalities and all animals showed expected gains in bodyweight. Decreased respiratory rate was noted 1 and 2 d after dosing in the initial treated animal but there were no signs of toxicity noted in the remaining animals. At necropsy a cavity in the right kidney, which was considered to be a genetic defect and not test item related, was noted in 1 animal. No abnormalities were noted at necropsy of the remaining animals. Therefore, the acute oral median lethal dose (LD50) of the test item was estimated to be greater than 2000 mg/kg bodyweight.

Acute toxicity via the inhalation route: An acute inhalation study was performed in the rat in accordance with GLP and OECD Guideline 436. A group of 6 RccHanTM : WIST strain rats (3 males and 3 females) was exposed to an aerosol atmosphere of the test item with a mean achieved atmosphere concentration of 5.92 mg/L for 4 h using a nose only exposure system, followed by a 14 d observation period. There were no mortalities and no macroscopic abnormalities were detected amongst animals at necropsy. Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur. Animals recovered quickly to appear normal from Days 5 to 7 post-exposure. All male animals and 1 female exhibited slight bodyweight losses or showed no bodyweight gain on the first day post-exposure. Bodyweight gains were noted in all animals during the remainder of the recovery period, with the exception of 1 female animal which exhibited a bodyweight loss from Days 1 to 3 and showed no bodyweight gain from Days 3 to 7 post-exposure. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of the test item was greater than 5.92 mg/L.


Justification for selection of acute toxicity – oral endpoint
The study is a GLP guideline study and is well documented and has been assigned a reliability 1.

Justification for selection of acute toxicity – inhalation endpoint
The study is a GLP guideline study and is well documented and has been assigned a reliability 1.

Justification for classification or non-classification

Acute oral toxicity: The available study has been assigned reliability 1 and is considered as acceptable for classification.The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight. As such, the test item can be considered to be non-classified.

Acute inhalation toxicity: The available study has been assigned reliability 1 and is considered as acceptable for classification.The acute inhalation median lethal concentration (4 h LC50) of the test item in the RCCHan™:WIST strain rat was greater than 5.92 mg/L. As such the test item can be considered to be non-classified.