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Description of key information

Acute toxicity after single oral application was tested in female rats, which received up to 300 mg/kg bw (three females) or 2000 mg/kg bw/d (two groups, each of three females). All animals survived until the end of the study without showing any signs of toxicity. Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain. At necropsy, no macroscopic findings were observed. The LD50 value for acute oral toxicity is > 2000 mg/kg bw.
A single dermal application of the test item to 10 rats (5 males and 5 females) at a dose of 2000 mg/kg bw was associated with no mortality and neither signs of irritation. The dermal LD50 was determined to be > 2000 mg/kg bw
The study was carried out according to OECD guideline 436. The inhalatory LC50 value of the test item in Wistar rats was established to exceed 5 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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed Guideline study according to 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
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0815)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept in groups / individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions
Route of administration:
oral: gavage
Vehicle:
physiological saline
Details on oral exposure:
The animals were marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test item was administered. Food was provided again approximately 4 hours post dosing.
The test item was administered at a single dose by gavage using a feeding tube.
The test item was administered at a dose volume of 10 mL/kg body weight.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
The starting dose was selected to be 300 mg/kg body weight. As no compound-related mortality was recorded for any animal of step 1,
it was decided for animal welfare reason to directly move on to a dose of 2000 mg/kg body weight in a second step.
Based on these results and according to the acute toxic class method regime, a third step was performed at a dose of 2000 mg/kg body weight.
Based on these results and according to the acute toxic class method regime no further testing was required.

Observation Period
All animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality.

Weight Assessment
The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.

Clinical Examination
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded.
Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined.
Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected
intraperitoneally (Narcoren®, Merial; lot no.: 218121; expiry date: 12/2014) at a dosage of approximately 8 mL/kg bw.
All animals were subjected to gross necropsy. All gross pathological changes were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation. The preserved tissues of which no histopathological evaluation was made will be discarded 3 months after the release of the final report unless otherwise agreed upon with the sponsor.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results,
a statistical evaluation of the results is not regarded as necessary.
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals survived until the end of the study without showing any signs of toxicity.
Clinical signs:
All animals survived until the end of the study without showing any signs of toxicity.
Body weight:
Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.
Gross pathology:
At necropsy, no macroscopic findings were observed in any animal of any step.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present study, a single oral application of the substance to rats at a dose of 2000 mg/kg body weight was associated with no signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 > 2000 mg/kg body weight.
Executive summary:

Under the conditions of the present study, a single oral application of the substance to rats at a dose of 2000 mg/kg body weight was associated with no signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): unclassified In conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC the test item has no obligatory labelling requirement for toxicity. According to Annex I of Regulation (EC) 1272/2008 the test item has no obligatory labelling requirement for toxicity and is not classified. According to GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for toxicity and is not classified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
1 (reliable without restriction)

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:
Jan - Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: appox. 11 weeks
- Weight at study initiation: 309 g (males), 194 g (females)
- Housing: three animals per Makrolon type IV cage
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Germany), ad libitum except during exposure to the test substance
- Water (e.g. ad libitum): tap water, ad libitum except during exposure to the test substance
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jan To: 3 Feb 2014
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
- Exposure chamber
The design of the exposure chamber is based on the flow past nose-only inhalation chamber. The chamber consisted of three animal sections with eight animal ports each. Each animal port had its own atmosphere inlet and exhaust outlet. The animals were placed in restraining tubes and connected to the animal ports. The number of animal sections and number of open inlets were adopted to the air flow in such way that at each animal port the theoretical air flow was at least 1 L/min, which ensures an adequate oxygen supply to the animals. The main inlet of the test atmosphere was located at the top section and the main outlet was located at the bottom section. The direction of the flow of the test atmosphere guaranteed a freshly generated atmosphere for each individual animal. All components of the exposure chamber in contact with the test material were made of stainless steel, glass, rubber or plastic. To avoid exposure of the personnel and contamination of the laboratory the exposure chamber was placed in a fume hood, which maintained at a slight negative pressure.

- Test atmosphere generation
Administering the test substance to a stream of pressurized air using a combnation of a durst feeder and air mover generated an aerosol. The aerosol was passed through the exposure chamber. The mean total airflow was 27 L/min. From the exposure chamber the test atmosphere was passed through a filter before it was released to the exhaust of the fume hood.

- Test atmosphere characterisation

(A) Nominal concentration
The nominal concnetration was caculated by dividing the amount of test substance used by the volume of pressurized air (average air flow times exposure time) entering the exposure chamber used for exposure of the animals. Due to the small volume of the exposure chamber the equilibrium time was negligible. The volmue of air was calculated from the average air flow (measured by means of thermal mass flow meters and was recorded regularly, preferably in 30 min intervals) and the exposure time.

(B) Actual concentration
The actual concentration was determined 25 times during the exposure period. Samples were drawn from the test atmosphere through a tube in one of the free animal ports of the middle section of the exposure chamber. Samples were drawn through a glass fiber filter. The collected amount of the test substance in the air sample was measured gravimetrically. Sample volumes were measured by means of a dry gas meter.
Subsequently the time-weighted mean concnetration with the standard deviation was calculated.

(C) Particle size characterization
The particle size distribution was characterized twice during the exposure period. The samples were drawn (2 L/min) from the test atmosphere through a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters amd a fiber glass back-up filter were measured gravimetrically. Subsequently the Mass Median Aerodynamic Diameter (MMAD) and the Geometric Standard Deviation (GSD) were determined.

(D) Stability monitoring
It was considered that the opacity of the test atmosphere could not be reliably monitored by means of an aerosol monitoring system. An indication of the stability of the test atmosphee was obtained from the concentration measurements, which were equally distributed over time.

(E) Temperature and relative humidity
The temperature and relative humidity were measured with a humidity and temperature indicator and were recorded after the animals were placed in the experimental set-up and at 30 min intervals after initiation of the exposure. The probe was inserted in a tube mounted in one of the free animal ports of the middle section of the exposure chamber. The temperature of the atmosphere was between 20.9 and 21.0°C and relative humidity was between 28 and 32%. These conditions were considered appropriated for this relatively short 4 hours exposure duration.
















Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5 mg/L (target concentration)
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Viability: twice daily
Clinical signs: three times during exposure for mortality, behavioural signs of distress and effects on respiration (during exposure); on day 1, one and three hours after exposure and once daily thereafter until day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration recorded (after exposure)
Body weights: days 1 (pre-administration), 2, 4, 8 and 15
- Necropsy of survivors performed: yes, all animals assigned to the study were subjected to necropsy and description of all internal macroscopic abnormalities were recorded. Particular attention was given to any changes in the respiratory tract.
Statistics:
not mandatory
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.1 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality occured.
Clinical signs:
other: During exposure, slow and shallow breathing was seen in all animals. After exposure no clinical signs of systemic toxicity were noted.
Body weight:
The body weight gain in all males and one female was within the range expected for rats of this strain and age used in this type of study. Two females showed body weight loss during the first week and one female did not gain weight during the second week while the other did gain weight.
Gross pathology:
No abnormalities were found at macroscopic post mortem exmination of the animals.
Other findings:
none
Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
The inhalatory LC50 value of the test item in Wistar rats was established to exeed 5 mg/L. Based on these results the test substance does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity.
Executive summary:

Assessment of acute inhalation toxicity in the rat (acute toxic class method)

The study was carried out according to OECD guideline 436. The test substance was administered as an aerosol by inhalation for 4 hours to one group of three male and three female Wistar rats. Animals were subjected to dauly observations and determination of body weights on days 1, 2, 4, 8 and 15. Macroscopic examination was performed after terminal sacrifice (day 15). The mean actual concentration was 5.1 +/- 0.13 mg/L. The nominal concentration was 15.6 mg/L. The generation efficiency (ratio of actual and nominal concentraion) was 33%. The concentration measurements equally distributed over time showed that the substance was sufficiently stable. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice. The MMAD was 3.4 µm (gsd 2.1) and 3.5 µm (gsd 2.1).

No mortaliy occurred. During exposure, slow and shallow breathing was seen in all animals. After exposure, no clinical signs of systemic toxicity were noted. The body weight gain in all males and one female was within the range expected for rats of this strain and age used in this type of study. Two females showed weight loss during the first week and one female did not gain weight during the second week while the other did gain weight. No abnormalities were found at macroscopic post mortem examination of all animals.

The inhalatory LC50, 4h value in Wistar rats was established to exceed 5.1 mg/L.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5.1
Quality of whole database:
1 (reliable without restriction)

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:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well reported guideline study according to 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
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:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1114)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8
(drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions
Type of coverage:
semiocclusive
Details on dermal exposure:
Preparation of the Animals:
The animals were marked for individual identification by tail painting.
Approximately 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper.
Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.
Application:
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period.
The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.
Duration of exposure:
The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed using aqua ad injectionem.
Doses:
The test item was applied at a single dose of 2000 mg/kg body weight to each animal.
No. of animals per sex per dose:
5 male and 5 female
Control animals:
not required
Details on study design:
Observation period:
All animals were observed for 14 days after dosing

Weight Assessment:
The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

Clinical Examination:
careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes
and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded.
Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes.
Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined.
Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology:
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally
(Narcoren®, Merial) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy.
All gross pathological changes were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation.
The preserved tissues of which no histopathological evaluation was made will be discarded 3 months after the release of the final report
unless otherwise agreed upon with the sponsor.

Evaluation of Results:
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.
On the basis of the test results, the test item may be classified in one of the following classes in conformity with the criteria given in
Annex VI to Commission Directive 2001/59/EC:
• Very toxic
Substances and preparations shall be classified as very toxic, and assigned the symbol “T+” and indication of danger “very toxic”
in accordance with the criteria specified below:
R27 Very toxic in contact with skin
- LD50 dermal, rat or rabbit: <= 50 mg/kg
• Toxic
Substances and preparations shall be classified as toxic, and assigned the symbol “T” and indication of danger “toxic”
in accordance with the criteria specified below. Risk phrases shall be assigned in accordance with the following criteria:
R24 Toxic in contact with skin
- LD50 dermal, rat or rabbit: 50 < LD50 <= 400 mg/kg
• Harmful
Substances and preparations shall be classified as harmful, and assigned the symbol “Xn” and indication of danger
“harmful” in accordance with the criteria specified below. Risk phrases shall be assigned in accordance with the following criteria:
R21 Harmful in contact with skin
- LD50 dermal, rat or rabbit: 400 < LD50 <= 2000 mg/kg
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in
Annex I of Regulation (EC) 1272/2008:
Category 1: LD50 <= 50 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 2: LD50 > 50 mg/kg <= 200 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 3: LD50 > 200 mg/kg <= 1000 mg/kg. DANGER. Skull and crossbones in diamond. Toxic in contact with skin.
Category 4: LD50 > 1000 mg/kg <= 2000 mg/kg. WARNING. Exclamation point in diamond. Harmful in contact with skin.
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in
GHS - Globally Harmonized System of Classification and Labelling of Chemicals, third revised edition, July 2009:
Category 1: LD50 <= 50 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 2: LD50 > 50 mg/kg <= 200 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 3: LD50 > 200 mg/kg <= 1000 mg/kg. DANGER. Skull and crossbones in diamond. Toxic in contact with skin.
Category 4: LD50 > 1000 mg/kg <= 2000 mg/kg. WARNING. Exclamation point in diamond. Harmful in contact with skin.
Category 5: LD50 > 2000 mg/kg <= 5000 mg/kg. WARNING. No symbol. May be harmful in contact with skin.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results,
a statistical evaluation of the results is not regarded as necessary.
Sex:
male/female
Dose descriptor:
approximate LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
No mortality was observed.
Clinical signs:
No treatment-related effects were observed.
Body weight:
A slight weight loss was recorded for 1 out of 5 female animals during the first week, but all of the female animals showed weight gain
during the second week. The effects on weight development might be secondary to the dressing, and toxicological relevance
of this finding cannot clearly be concluded.
The male animals showed weight gain during the first and the second week of the observation.
Gross pathology:
No treatment-related effects were observed.
Other findings:
No treatment-related effects were observed.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation.The dermal LD50 was determined to be > 2000 mg / kg body weight.
Executive summary:

Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation.The dermal LD50 was determined to be > 2000 mg / kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
1 (reliable without restriction)

Additional information

Based on the results of an oral toxicity study, an acute dermal toxicity study and an acute inhalation toxicity study the LD50values for acute oral and dermal toxicity was determined to be > 2000 mg/kg bw and the LC50 > 5.1 mg/L.


Justification for selection of acute toxicity – oral endpoint
GLP compliant guideline study.

Justification for selection of acute toxicity – inhalation endpoint
GLP compliant guideline study.

Justification for selection of acute toxicity – dermal endpoint
GLP compliant guideline study.

Justification for classification or non-classification

Due to the results described in the acute oral and dermal toxicity studies (LD50oral/dermal in rats > 2000 mg/kg bw; LC50 > 5.1 mg/L) the substance does not have to be classified as acute orally, dermally or inhalatory toxic.

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