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Administrative data

Description of key information

Acute toxicity - Oral: In an acute oral toxicity study in female Sprague-Dawley rats, following the up-and-down procedure in accordance with the OECD Guideline 425, the LD50 was established to be greater than 2000 mg/kg (Vasquez, 2012).

Acute toxicity - Inhalation : In an acute inhalation toxicity study in male and female Wistar rats, following a method according to (US EPA) guidelines and OPPTS 870.1303, the LC50 was established to exceed 15.246 mg/L ( Ferreira, 2012).

Acute toxicity - Dermal: In an acute dermal toxicity study in male and female Sprague-Dawley rats, following the standard acute method according to OECD Guideline 402 and EC method B.3, the LD50 was established to exceed 2000 mg/kg body weight (Vasquez, 2012).

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:
12 December 2010 - 6 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Version / remarks:
3 October 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
GLP compliance:
yes
Test type:
up-and-down procedure
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
The identity, purity, strength and stability of the test article were the responsibility of the Sponsor.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test article was dosed as received

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 8-10 weeks at start of dosing
- Fasting : yes, animals were fasted overnight prior to dose administration. Food was returned to the animals ~ 30 minutes after dosing.
- Weight at study initiation: 185-199 grams
- Fasting period before study: yes
- Housing: Animals were group housed by sex upon receipt and individually housed upon assigment to study in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals". No other species were kept in the same room.
- Diet (e.g. ad libitum): Harlan Teklad Rodent Diet (certified)
- Water (e.g. ad libitum): Tap water was available ad libitum
- Acclimation period: Minimum of five (5) days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 23
- Humidity (%): 17 to 54%.
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The dose volumes were 0.54 and 1.98 mL/kg and were based on the relative density of 1.01 g/mL as per MSDS.

Method of study performance:
The first animal was dosed at an initial dose level of 550 mg/kg. Since this animal survived, the second animal received a higher dose (2000 mg/kg). Two additional animals were dosed at 2000 mg/kg. The dose for each successive animal was adjusted up or down, depending on the previous result. The test continued based on the fixed time interval outcomes of all the animals up to that point and until one of the stopping criteria was first met. There were three possible stopping criteria.
• 3 consecutive animals survive at the limit dose;
• 5 reversals occur in any 6 consecutive animals test;
• or at least 4 animals have followed the first reversal and the specific likelihood-ratios exceed the critical value.
Dose progression and stopping criteria were calculated using a dedicated software program [Acute Oral Toxicity (Guideline 425) Statistical Program, Version 1.0] provided by the EPA.
Doses:
550 and 2000 mg/kg
No. of animals per sex per dose:
4 females in total
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Morbidity: manually recorded once daily
Clinical observations: on the day of dosing (Day 1) animals were observed prior to dosing and approximately 30 minutes and 4 hours post-dose. Animals were observed once daily thereafter (Days 2-15).
Body weights - at study initiation (Day 1), Days 8 and 15
- Necropsy of survivors performed: yes
All surviving rats were sacrificed by CO2 inhalation and a gross necropsy was performed at termination of the study
The necropsy included examination of:
• the external body surface
• all orifices
• the cranial, thoracic and abdominal cavities and their contents.
Statistics:
The LD50 was calculated for the main test (when the data allow) using the AOT425StatPgm developed by Westat May, 2001 (available via EPA website).
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Mortality was not observed in any of the animals dosed at 550 or 2000 mg/kg
Clinical signs:
All animals appeared normal throughout the study at 550 and 2000 mg/kg
Body weight:
No biologically significant effect was seen on body weights of animals on Day 8 and 15.
Gross pathology:
Terminal necropsy revealed no visible lesions in any of the animals at 550 or 2000 mg/kg.
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
Based on the results of this study, the oral LD50 for the test substance in rats was estimated to be greater than 2000 mg/kg and hence a Risk Phrase Not Required by EEC and a category 5 according to GHS.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw

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:
06 September 2011 to 06 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8684-90, Experimental

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRl's (DF) rearing house
- Age at study initiation: 10 weeks
- Weight at study initiation: 305-347 grams
- Fasting period before study: no
- Housing: 41x34x20 cm polypropylene cages with autoclaved wood shavings and stainless steel mesh lids containing five rats of same sex per cage on the experimental phase were used
- Diet (e.g. ad libitum): Pelleted and autoclaved commercial diet for rats
- Water (e.g. ad libitum): Filtered potable water
- Acclimation period: 14 days prior to exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 to 21.8°C
- Humidity (%): 37.9 to 69.8%
- Air changes (per hr): 10 to 20 air changes in the room per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chamber used was a Jaeger Inhalation Chamber, Mark II with a collision nebulizer integrated system
- Exposure chamber volume: 667 ml/min/animal
- Method of holding animals in test chamber: Rats were exposed to the aerosolized test item in plexiglass nose-only tubes applying a direct-flow exposure principle.
- Source and rate of air: The source of air used came from an air compressor
- Method of conditioning air: The compressed air was treated with a filter absolute model F - 771 K03 Trox and filter of activated carbon F - 760 Trox;
- System of generating particulates/aerosols: collision nebulizer integrated system
- Method of particle size determination: Two equally time-spaced air samples were taken from the vicinity of the breathing zone using an air sampling pump previously calibrated to a 0.500 L/min flow rate, to collect the aerosol in a Seven Stage Cascade Impactor (ln-Tox, Albuquerque, New Mexico, U.S.A.) for five minutes. Before and after sampling, the stages were weighed
- Treatment of exhaust air:
- Temperature, humidity, pressure in air chamber: The study animals were maintained at a temperature from 22.3 to 23.8°C and a relative humidity from 51 to 59%. Temperature and relative humidity were recorded four times during the exposure. The equipment used to measure temperature and humidity was a digital thermo-hygrometer TFA;

TEST ATMOSPHERE
- Brief description of analytical method used: Rats were exposed to the aerosolized test item in plexiglass nose-only tubes applying a direct-flow exposure principle.
- Samples taken from breathing zone: yes/no

VEHICLE
- Composition of vehicle (if applicable):
- Concentration of test material in vehicle (if applicable):
- Justification of choice of vehicle:
- Lot/batch no. (if required):
- Purity:

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution:
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration:
Duration of exposure:
4 h
No. of animals per sex per dose:
five/sex/cage
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Aerodynamic particle-size distribution: Before and after sampling, the stages were weighed
- Mortality: yes
- Necropsy of survivors performed: yes. All exposed rats were necropsied and subsequently incinerated. The presence or absence of any macroscopic finding observed on gross necropsy was recorded systematically and individually
- Clinical examinations: Following exposure, two clinical observations were made during the first 24 hours and then daily during a fourteen­ day observation period
Statistics:
Not applicable
Preliminary study:
Preliminary test without animals was conducted to define the appropriate conditions to reach a test atmosphere with adequate particle-size.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
>= 15.246 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No compound-related deaths were observed during this study.
Clinical signs:
The compound-related clinical signs observed in this study were: piloerection, slight apathy, kyphosis, slight dyspnea, tremors, epistaxis and chromodacryorrhea. These were acute systemic signs that started on days 0 and 1 and reverted on days 1 to 3 of the observation period.
Body weight:
The mean body weight increased for both sexes in all post-exposure weighings, except in the first post-exposure weighing. All animals exceeded their initial body weight at the end of the observation period of 14 days.
Gross pathology:
No compound-related macroscopic findings were observed during this study.
Other findings:
Histopathology
It was not considered necessary due to the reversibility of the clinical signs and to the absence of compound-related macroscopic findings.
Interpretation of results:
GHS criteria not met
Conclusions:
The combined (males and females) median lethal concentration in a 4-hour nose-only exposure period (4-h LC50) to the test substance aerosol inhaled by Wistar Hannover rats was greater than 15.246 mg/L.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
15.246 mg/m³

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:
12 December 2010 - 27 December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-30°C)
- Stability under test conditions: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dosed neat as received from the sponsor.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 7-8 weeks
Protocol deviation:
The protocol stated the age range for the animals was 8-24 weeks. The actual age of the male animals on study was 7 weeks. This deviation of one week was not of a magnitude to have had an impact on the outcome or integrity of the study since the animals were still considered young adults and were within the weight range of the protocol.
- Weight at study initiation: 190-226 grams
- Housing: Animals were group housed by sex upon receipt and individually housed upon assignment to study in compliance with the National Research Council "Guide for the Care and Use of Laboratory Animals." Calvert is a USDA registered and fully AAALAC accredited facility. The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Rodent Diet (certified) ad libitum, as per Calvert SOP
- Water (e.g. ad libitum): tap water was available ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of five days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 22°C
- Humidity (%): 18 to 52%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Protocol deviations:
The temperature and relative humidity recorded in the animal room were not within the protocol limits of 22 ± 3°C and 30 to 70% on several occations during the course of the study. The ranges were 16 to 22°C and 18 to 52%, respectively. These deviations had no impact on the study outcome since the animals were observed daily and were considered to be normal.
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal area of the trunk
- Type of wrap if used: The test article was applied on an intact skin site for each animal, covered with a gauze patch/dental dam, wrapped with an elastic bandage and secured with non-irritating tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After exposure the site was unwrapped and wiped with water or mineral oil and gauze to prevent ingestion.
- Time after start of exposure: twenty-four (±0.5) hours.

TEST MATERIAL
One group of animals received the test article at 2000 mg/kg, 1.98 mL/kg. Since no mortality occurred in the Limit Test, a full Definitve Test was not necessary.
Duration of exposure:
The test article was applied once and remains in contact with the skin site for twenty-four (±0.5) hours.
Doses:
2000 mg/kg
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/morbidity: once daily;
Clinical observations: Clinical observations were recorded immediately after unwrap from the 24 hour exposure (Day 2) and daily thereafter through Day 15. At the time of clinical observations, the dose site was also evaluated for dermal irritation.
Body weight: Animals were weighed prior to dosing on Day 1 and on Days 8 and 15.
- Necropsy of survivors performed: yes,
All animals were euthanized by CO2 asphyxiation.
The necropsy included examination of the external body surface, all orifices, the thoracic, abdominal and pelvic cavities and their contents
Statistics:
Body weights were summarized using descriptive statistics (mean and standard deviation).
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Mortality was not observed in any of the animals at 2000 mg/kg.
Clinical signs:
Abnormal gait and stance was observed immediately after unwrap from the 24 hour exposure (Day 2) of the study. No other abnormal clinical signs were observed during the study. No erythema or edema was observed at the application sites during the study.
Body weight:
No biologically significant effect was seen on body weight in the animals on Days 8 and 15. All of the animals gained weight during the course of the study.
Gross pathology:
No visible lesions were observed in any of the animals at terminal necropsy.
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
Based upon the results of the Acute Dermal Toxicity Study in Rats with the test substance, the estimated LD50 was considered to be greater than 2000 mg/kg body weight. Therefore, according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS), the test substance was considered to be Category 5 test article. An EEC Risk Phrase is not required and it is Not WHMIS Controlled.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw

Additional information

Acute toxicity: Oral

An acute oral toxicity study with the test item according to up-and-down procedure in female Sprague-Dawley rats (OECD guideline 425) was performed in order to assess the toxicity of the test article following a single oral dose to the rat (Vasquez, 2012).

Initially, one female Sprague Dawley rat was dosed at 550 mg/kg. No mortality was observed at 550 mg/kg and dosing continued in another female at 2000 mg/kg. Dosing continued in two additional females al 2000 mg/kg as per protocol guidelines. A total of 4 females were dosed. Mortality checks were made once daily. Clinical observations were recorded prior to dosing, as well as at 30 minutes, 4 hours post-dose, and daily thereafter through Day 15. Body weights were recorded on the day of dosing (Day 1), and on Days 8 and 15. All rats were euthanized by CO2 asphyxiation and necropsied on Day 15.

For doses of 550 and 2000 mg/kg, no mortality was observed. All animals appeared normal throughout the study al 550 and 2000 mg/kg. No biologically significant effect was seen on body weights on Days 8 or 15. Terminal necropsy revealed no visible lesions in any of the animals at 550 or 2000 mg/kg.

Based on the results of the study, the oral LD50 for the test substance in rats was estimated to be greater than 2000 mg/kg.

Acute toxicity: Inhalation

An acute inhalation toxicity study with the test item in male and female Wistar rats was performed in order to assess the toxicity of the test article following a exposure period of 4 hours (Ferreira, 2012). One group of rats (five/sex) was nose-only exposed to the undiluted aerosolized test item for an exposure period of 4 hours, using a total air flow of 8L/min.The analysis of the aerodynamic particle-size distribution indicated that 47.67 to 50.53% of the mass collected from the aerosol generated was within the respirable diameter (< 3.42 µm). The Mass Median Aerodynamic Diameter (MMAD) ranged from 3.91 to 3.97 µm and the Geometric Standard Deviation (GSD) ranged from 1.97 to 2.02. The mean actual concentration tested was 15.246 mg/L. Neither compound-related deaths nor compound-related macroscopic findings on gross necropsy were observed in the study. The compound-related clinical signs observed during the 14-day observation period were: piloerection, slight apathy, kyphosis,slight dyspnea, tremors, epistaxis and chromodacryorrhea. These were acute systemic signs, which started on days 0 and 1 and reverted to normal on days 1 to 3 of the observation period. The mean body weight increased for both sexes in all post-exposure weighings, except in the first post-exposure weighing. All animals exceeded their initial body weight at the end of the observation period of 14days. Therefore, the combined (males and females) median lethal concentration in a 4-hour nose-only exposure period (4-h LC50) to aerosolized test substance inhaled by Wistar Hannover rats was greater than 15.246 mg/L.

Acute toxicity: Dermal

An acute dermal toxicity study with the test substance according to the standard acute method in male and female Sprague-Dawley rats (OECD guideline 402 and EU Method B.3) was performed in order to evaluate the potential toxicity or to determine the median lethal dose (LD50) of a test article following a single administration via the dermal route (Vasquez, 2012)

One group of ten Sprague-Dawley rats (five males and five females) received the test article dermally at a dose level of 2000 mg/kg. Mortality checks were made once daily. Clinical observations and dermal irritation were recorded immediately post-unwrap, and daily thereafter through Day 15. Body weights were recorded on the day of dosing (Day 1), and on Days 8 and 15. All rats were euthanized by CO2 asphyxiation and necropsied on Day 15.

Abnormal gait and stance was observed immediately after unwrap from the 24 hour exposure (Day 2) of the study. No other abnormal clinical signs were observed during the study. No erythema or edema was observed at the application sites during the study. No mortality was observed during the study. No biologically significant effect was seen on body weight in the animals on Days 8 and 15. No visible lesions were observed in any of the animals at terminal necropsy. Based upon the results of the Acute Dermal Toxicity Study in Rats with the test substance, the estimated LD50 was considered to be greater than 2000 mg/kg body weight.

 

 

Justification for classification or non-classification

The oral LD50 for the test substance was estimated to be greater than 2000 mg/kg. According to the criteria in the CLP Regulation, the test substance should not be classified for acute oral toxicity.

The 4-h LC50 for the test substance was determined to be greater than 15.246 mg/L. According to the criteria in the CLP Regulation, the test substance should not be classified for acute inhalation toxicity.

The dermal LD50 for the test substance was estimated to be greater than 2000 mg/kg. According to the criteria in the CLP Regulation, the test substance should not be classified for acute dermal toxicity.