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Description of key information

The acute toxicity study by oral route was conducted on the registered substance according to OECD Testing 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 body weight. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The acute toxicity study by inhalation route was conducted on the registered substance according to OECD Testing Guideline 403. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 5 mg/l (air). The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

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:
From 18 August 2015 to 17 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 420. The study was conducted on the registered substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatization period of at least five days the animals were selected at random and given a unique number within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The body weight variation did not exceed ±20% of the body weight at the start of treatment.
The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with
wood flakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would 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 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any
contaminant of a level that might have affected the purpose or integrity of the study.
Rats are the preferred species of choice as they are historically used for safety evaluation studies and are specified in the appropriate test guidelines.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
For the purpose of the study, the item was freshly prepared, as required, as a suspension in distilled water.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Doses:
- 300 mg/kg
- 2000 mg/kg
No. of animals per sex per dose:
1 animal received a dose of 300 mg/kg, 5 animals received a dose of 2000 mg/kg
Control animals:
no
Details on study design:
Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily.
Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
Preliminary study:
Dose level 300 mg/kg: No mortality. No signs of systemic toxicity were noted during the observation period. The animal showed expected gains in body weight over the observation period. No abnormalities were noted at necropsy.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths at either dose level
Clinical signs:
No signs of systemic toxicity were noted during the observation period at either dose level
Body weight:
All animals showed expected gains in body weight over the observation period at either dose level
Gross pathology:
No abnormalities were noted at necropsy.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
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 body weight. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The acute toxicity: oral of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 420. Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. At dose level 300 mg/kg there was no mortality, no signs of systemic toxicity noted during the observation period, the animal showed expected gains in body weight over the observation period and no abnormalities were noted at necropsy. Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated. At dose level 2000 mg/kg there was no mortality, all animals showed expected gains in body weight and no abnormalities were noted at necropsy. 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 body weight. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The study was considered as fully reliable as it was conducted on the registered substance, as defined in section 1.1.

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:
From 7 August 2015 to 8 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 403. The study was conducted on the registered substance.
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
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, 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, Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water, bedding, chew blocks and cardboard “fun tunnels” 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.
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Atmosphere Generation
A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected 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 SAG 410. The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410. 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 in an attempt 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 100 % of target and only three deaths occurred, no further levels were required.

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.

Exposure Chamber Atmosphere Concentration
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum
pump.
Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
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 812 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was difficult.

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 (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 μm cut points) with stainless steel collection substrates and a backup glass fiber 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 cutoff point size. In this way, the proportion (%) of aerosol less than 8.4, 7.3, 3.6, 1.3, 0.94 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:
5.02 mg/l
No. of animals per sex per dose:
five males and five females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 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
- Other examinations performed: clinical signs, body weight
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 2 000 mg/L air
Mortality:
Two male rats out of five and one female rat out of five died.
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
Body weight:
Three males and three surviving female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. With the exception of one surviving female animal which exhibited a further body weight loss from Days 1 to 3 post-exposure, all surviving animals exhibited body weight gains during the remainder of the recovery period.
Gross pathology:
With the exception of two instances of dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period.
Abnormally dark lungs were noted at necropsy in the three animals that died during the course of the study.
Due to the clinical observations noted and macroscopic abnormalities detected, the deaths noted during the study may have been attributable to local toxicity.

The mortality data are summarized as follows:

   Mean Achieved Atmosphere Concentration (mg/l)         Deaths
 Male  Female Total 
5.02   2/5  1/5  3/10
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 5 mg/l (air). The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The acute toxicity: inhalation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 403. A group of ten RccHan™ : WIST strain rats (five males and five females) was exposed to a dust atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Three deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.02 mg/L for four hours. Decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection and wet fur were observed during the study; surviving animals recovered to appear normal on Day 5 post-exposure. Three males and three surviving female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. With the exception of two instances of dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period. Abnormally dark lungs were noted at necropsy in the three animals that died during the course of the study.

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 5.02 mg/l. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
5 000 mg/m³
Quality of whole database:
The study was considered as fully reliable as it was conducted on the registered substance, as defined in section 1.1.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
According to Moore & al. (2013), oral administration represents a worst-case scenario compared to dermal administration, and a substance with a LD50 (oral) >2000 mg/kg is not expected to be classified for acute systemic toxicity by the dermal route. Moreover, in accordance with Annex VIII of REACH, Column 2, assessing the acute toxicity by dermal route is relevant only if the inhalation of the substance is unlikely, and if the physicochemical and toxicological properties suggest potential for a significant rate of absorption through the skin, which is not the case for the registered substance as the substance is a solid generating dust when handle.

In addition due to the particle size of the substance, it cannot be absorbed as such following a dermal exposure. It must first be dissolved for the absorption process to occur. However, the high lattice energy of the structure implies that it would require conditions far more extreme than those the substance will experience on the skin. Therefore no dissolution is expected following a dermal exposure, and no absorption will occur.

Moore NP, Andrew DJ, Bjerke DL, Creton S, Dreher D, Holmes T, Prieto P, Seidle T, Rowan TG (2013) Can acute dermal systemic toxicity tests be replaced with oral tests? A comparison of route-specific systemic toxicity and hazard classifications under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Regul Toxicol Pharmacol. 2013 Jun;66(1):30-7
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The acute toxicity: oral of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 420. Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. At dose level 300 mg/kg there was no mortality, no signs of systemic toxicity noted during the observation period, the animal showed expected gains in body weight over the observation period and no abnormalities were noted at necropsy. Based on the results at a dose level of 300 mg/kg, a dose level of 2000 mg/kg body weight was investigated. At dose level 2000 mg/kg there was no mortality, all animals showed expected gains in body weight and no abnormalities were noted at necropsy. 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 body weight. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The acute toxicity: inhalation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 403. A group of ten RccHan™ : WIST strain rats (five males and five females) was exposed to a dust atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. Three deaths occurred in a group of ten rats exposed to a mean achieved atmosphere concentration of 5.02 mg/L for four hours. Decreased respiratory rate, increased respiratory rate, hunched posture, pilo-erection and wet fur were observed during the study; surviving animals recovered to appear normal on Day 5 post-exposure. Three males and three surviving female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. With the exception of two instances of dark patches on the lungs, no macroscopic abnormalities were detected at necropsy amongst animals that survived until the end of the fourteen day recovery period. Abnormally dark lungs were noted at necropsy in the three animals that died during the course of the study. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 5.02 mg/l. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of acute toxicity – oral endpoint

This study was selected as the key study as it was a GLP study conducted on the registered substance according to OECD Testing Guideline 420. The LD50 was > 2000 mg/kg bw.

Justification for selection of acute toxicity – inhalation endpoint

This study was selected as the key study as it was a GLP study conducted on the registered substance according to OECD Testing Guideline 403. The LC50 was > 5000 mg/m3.

Justification for selection of acute toxicity – dermal endpoint

According to Moore & al. (2013), oral administration represents a worst-case scenario compared to dermal administration, and a substance with a LD50 (oral) >2000 mg/kg is not expected to be classified for acute systemic toxicity by the dermal route.

Moreover, in accordance with Annex VIII of REACH, Column 2, assessing the acute toxicity by dermal route is relevant only if the inhalation of the substance is unlikely, and if the physicochemical and toxicological properties suggest potential for a significant rate of absorption through the skin, which is not the case for the registered substance as the substance is a solid generating dust when handle.

Justification for classification or non-classification

The acute toxicity study by oral route was conducted on the registered substance according to OECD Testing 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 body weight. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The acute toxicity study by inhalation route was conducted on the registered substance according to OECD Testing Guideline 403. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 5 mg/l (air). The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.