Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Oral: OECD 423, rat (female), 2016: LD50 >2000 mg/kg bw
Dermal: OECD 402, rat (male/female), 2016: LD50 >2000 mg/kg bw (limit test)
Inhalation (dust): OECD 403, rat (male/female), 4 h exposure, 2016: 1.22 mg/L <
LC50 < 2.51 mg/L (1220 mg/m³ < LC50 < 2510 mg/m³)

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:
19 Mar - 12 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
Adopted in 2001
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA), Department of Science and Technology, Government of India
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:
TEST ANIMALS
- Source: Intox PVT. Ltd.
- Females nulliparous and non-pregnant: yes
- Age at start of treatment: 8 weeks
- Body weight at start of treatment: 145 - 155 g (females)
- Fasting period before study: Yes, rats were fasted overnight prior to dosing. Food was offered 3 - 4 h after dosing.
- Housing: group housing, 3 animals of the same dose group in one cage (cage size: 35 cm (L) x 22 cm (W) x 18 cm (H)), in sterilized solid bottom polypropylene cages with stainless steel grill tops and sterilized paddy husk bedding
- Diet: extruded pelleted rat feed Altromin (manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany, and supplied by ATNT Laboratories, Mumbai, India), ad libitum
- Water: filtered water of drinking water quality, ad libitum
- Acclimation period: 7 - 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 38 - 66
- Air changes (per hr): 11.03 - 14.6
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Mar 2016 To: 12 Apr 2016
Route of administration:
oral: gavage
Vehicle:
other: analytical grade water with 0.2% Tween 80
Details on oral exposure:
VEHICLE
- Test substance concentration in vehicle: 200 mg/mL
- Amount of vehicle: 10 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: Formulations of the test substance were prepared freshly before dosing on each day. The test substance was suspended in analytical grade water with 0.2% Tween 80 as a wetting agent.

CLASS METHOD
- Rationale for the selection of the starting dose: A previous acute toxicity study revealed that the oral LD50 value in male and female rats is >7080 mg/kg bw. Based on the available information, the present study was started at the dose level of 2000 mg/kg bw, according to OECD TG 423.


Doses:
2000 mg/kg bw (step 1 and step 2)
No. of animals per sex per dose:
3 female animals per group; two groups were tested stepwise in 2 consecutively steps (referred as step 1 and step 2)
Control animals:
no
Remarks:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for mortality twice a day throughout the observation period. Following test substance administration, animals were observed for clinical signs of toxicity 10 min, 30 min, 1 h, 2 h and 4 h post dosing and periodically thereafter for the first 24 h post dosing. During the 14-day observation period, animals were observed at least once a day. Body weight was measured at one day prior to dosing (Day 0), on the day of dosing (Day 1, fasting body weight), on Day 7 and at study termination on Day 15.
- Necropsy of survivors performed: yes
Statistics:
Body weight gain and group mean values were calculated.
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
No mortality occurred during the study period.
Clinical signs:
No clinical signs of toxicity were observed up to the end of the 14-day observation period.
Body weight:
No effect on body weight was noted.
Gross pathology:
Necropsy revealed no substance-related findings.

Table 1. Acute oral toxicity

 

Dose

[mg/kg bw]

Mortality

Clinical signs

 

N*

N*

Females

2000

0/6

0/6

*N = number of animals/ number of animals used (shown as combined values from step 1 and step 2)

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Conclusions:
In the present acute oral toxicity study in rats no mortality (0/6 rats) occured after single gavage of 2000 mg/kg bw of the test substance.
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Regulation (EC) No 1907/2006.

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:
10 Mar - 04 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
Adopted in 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
Adopted in 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
Adopted in 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
National Institute of Pharmacy and Nutrition, Budapest, Hungary
Test type:
traditional method
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Model and Services, Germany GmbH (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Sighting exposure (Group 0.1): 11 weeks; Main Study (Group 1): 8 weeks; Main Study (Group 2): 10 weeks
- Body weight at study initiation: Sighting exposure (Group 0.1): males: 416 g, females: 251 g; Main Study (Group 1): males: 317 - 331 g, females: 213 - 233 g; Main Study (Group 2): males: 361 - 396 g, females: 242 - 276 g
- Housing: sighting study: individually; main study: group housed, 5 animals, by sex per cage in polycarbonate solid floor cages (type II or III) with stainless steel mesh lids, Lignocel® Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH+Co. KG, Rosenberg, Germany), and cotton rolls as nest building material
- Diet: ssniff SM R/M “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: sighting study (Group 0.1): 28 days; main study (Group 1): 8 days; main study (Group 2): 20 days
- Microbiological status: SPF

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 – 25.8
- Humidity (%): 34 – 69
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Sighting exposure (Group 0.1): From: 10 Mar 2016 To: 24 Mar 2016; Main Study (Group 1): From: 01 Apr 2016 To: 15 Apr 2016; Main Study (Group 2): From: 20 Apr 2016 To: 04 May 2016
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 2.39 - <= 2.55 µm
Geometric standard deviation (GSD):
>= 2.81 - <= 2.92
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprised of 2 concentric anodised aluminium chambers.
- Exposure chamber volume: 3.85 L
- Method of holding animals in test chamber: polycarbonate restraint tubes located around the chamber
- Method of conditioning air: Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the appropriate dust generator.
- Rate of air (airflow): at least 0.5 L/min at each animal's exposure port (breathing zone)
- System of generating particulates/aerosols: Dust particles were produced in a dust generator according to Wright (TSE GmbH, Bad Homburg, Germany) located at the top of the exposure chamber. In this device a fixed blade scraped continuously the surface of the test substance compacted in a rotating reservoir. Dispersion was carried out by a high velocity air flow inside the outlet nozzle.
- Method of particle size determination: cascade impactor, 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany)
- Temperature and humidity in air chamber: 21.7 - 27 °C, 12 - 17.5%

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable volume of test atmosphere through weighed GF10 glass fibre filters (Whatman®, Whatman GmbH, Germany, Ref. no. 10370302). The difference in the pre and post sampling weights and divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration. Filter samples were collected at the breathing zone (approximately every 10 - 20 min) during each 4-h exposure period and analysed. The nominal concentration was calculated by dividing the mass of test substance disseminated into the chamber by the total volume of air that flow through the chamber during the same period.
- Samples taken from breathing zone: yes
- Actual time allowed for equilibrium of exposure concentration before animal exposure : 17 - 26 min

TEST ATMOSPHERE
- Particle size distribution: Sighting exposure (Group 0.1): 66.7% <4 µm; Main Study (Group 1): 69.0% <4 µm; Main Study (Group 2): 67.8% <4 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Sighting exposure (Group 0.1): 2.55 / 2.83; Main Study (Group 1): 2.39 / 2.81; Main Study (Group 2): 2.43 / 2.92

DOSE SELECTION RATIONALE
- Rationale for the selection of the starting concentration: In a dose-range finding study, sighting exposures using one male and one female animal were performed (Group 0.1). A target concentration of 2.5 mg/L was tested. Animals were exposed for 4 h to the test atmosphere. One female died after the exposure. Based on this lethality the main study was performed at target concentrations of 2.5 and 1.25 mg/L.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Sighting exposure (Group 0.1): 2.50 mg/L; Main Study (Group 1): 2.50 mg/L; Main Study (Group 2): 1.25 mg/L
No. of animals per sex per dose:
Sighting exposure (Group 0.1): 1 animal/sex/dose; Main Study (Group 1): 5 animals/sex/dose; Main Study (Group 2): 5 animals/sex/dose
Control animals:
no
Remarks:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were checked hourly during exposure, 1 h after exposure termination and twice daily during the 14-day observation period for morbidity/ mortality. Animals were observed for clinical signs at hourly intervals during exposure. Following exposure, clinical observations were performed twice on the day of exposure and subsequently once daily for 14 days. Individual body weights were recorded prior to treatment on the day of exposure (Day 0) and on Day 1, 3, 7 and 14, and prior to necropsy.
- Necropsy of survivors performed: yes
- Other examinations performed: Gross necropsy was performed with special attention to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Statistics:
For body weights, mean values were calculated for each sex.
Preliminary study:
The female animal of Group 0.1 died after the exposure on Day 0.
Following clinical signs of toxicity were observed: laboured respiration (slight to severe), clonic convulsion and decreased activity (slight) were observed in the female animal before its death. Laboured respiration (slight to severe), noisy respiration (moderate to severe), incoordination (slight to moderate), decreased activity (slight to moderate) and irritability were recorded in the surviving male animal. The male animal was symptom free from Day 7.
Slight body weight loss (9.4%) was observed in the surviving male animal after the exposure, which was followed by normal body weight gain.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1.22 - < 2.51 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
Group 1: 2/5 male rats died during the exposure, 1 male and 2 females after the exposure on Day 0, 2 female animals were found dead on Day 1, and 1 female on Day 2.
Group 2: 1/5 females died after the exposure on Day 0.
Clinical signs:
other: Laboured respiration was frequently observed. For further details see "Other findings".
Body weight:
Group 1: Slight to moderate body weight losses (7.9 and 10.4%) were recorded in the surviving males after the exposure. All surviving rats regained their initial body weight by Day 7 at the latest.
Group 2: Slight body weight losses (3.9 - 9.9%) were noted post exposure. From Day 3 onwards, all rats showed normal body weight gain.
Gross pathology:
Dark/red discoloration of the non-collapsed/collapsed lungs and white dry/powder material at the perinasal/perioral fur in all found dead animals, enlarged lungs and white mucoid material within trachea in 1/10 animal, and white creamy material observed in digestive content of the stomach in one female were regarded as test substance-related.
In surviving animals no test substance-related changes were noted at the end of the observation period on Day 14.
Other findings:
Clinical signs of toxicity:
Group 1: Laboured respiration (slight to severe) was recorded in the three males before their death. Laboured respiration (slight to severe), noisy respiration (slight), sneezing, activity decreased (slight to moderate), irritability were noted in the surviving two males. Laboured respiration (slight to severe), noisy respiration (slight), activity decreased (slight to moderate), irritability, tonic convulsion were recorded in the females before death. The surviving male animals were symptom free from Day 5 till study termination.
Group 2: Laboured respiration (slight to severe) was recorded in the female before its death. In the surviving animals, laboured respiration (slight to severe), noisy respiration (slight), sneezing, activity decreased (severe), irritability and tremor (intermittent) were observed. The animals were symptom free from Day 4 onwards.
Wet fur, ruffled fur or red-brown staining (as chromodacryorrhea) occurred in study animals from Day 0 up to Day 3. These signs were considered to be related to the restraint and exposure procedures or discomfort of the animals but not to be of toxicological relevance.

Table 2. Summary of acute inhalation toxicity

Group
Number /
Sex
Mean Achieved
Concentration
(mg/L)
Toxicological
Result*
Onset and Duration
of Toxicological
Clinical Signs (Day)
Occurrence of
Mortality
(Day)
Sighting exposure
0.1 / Male 2.53 0/1/1 0 - 6 -
0.1 / Female 2.53 1/1/1 0 0
Main study
1 / Male 2.51 3/5/5 0 - 4 0
1 / Female 2.51 5/5/5 0 - 1 0, 1, 2
2 / Male 1.22 0/5/5 0 - 1 -
2 / Female 1.22 1/5/5 0 - 1 0

* 1st = number of dead animals, 2nd = number of animals with clinical signs, 3rd = number of animals exposed

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the conditions of the present study, 8/10 animals (3/5 males and 5/5 females) were found dead after the exposure to 2.51 mg/L of the test substance for 4 h and 1/10 animals was found dead after the exposure to 1.22 mg/L of the test substance. The LC50 is considered to be between 1.22 mg/L and 2.51 mg/L.
Based on these results, the following classification under Regulation (EC) No 1272/2008 is justified: CLP: Acute Tox. 4, Inhalation, H332
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Regulation (EC) No 1907/2006.

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:
8 Mar - 29 Mar 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)
Version / remarks:
Current version adopted in 2017
Deviations:
yes
Remarks:
number and sex of test animals; males and females were used, 5/sex
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
Guideline in place during study conduct: adopted in 1987
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA), Department of Science and Technology, Government of India
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:
TEST ANIMALS
- Source: Intox PVT. Ltd., Pune, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Body weight range at study initiation: 207 - 252 g
- Housing: individually, in sterilised solid bottom polypropylene cages (sized: 35 cm (L) x 22 cm (W) x 18 cm (H)) with stainless steel grill tops and with bedding of clean and sterilised paddy husk
- Diet: extruded pelleted rat feed 'Altromin' (manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany and supplied by ATNT Laboratories, Mumbai, India), ad libitum
- Water: filtered and UV irradiated water in drinking water quality, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23
- Humidity (%): 42 - 66
- Air changes (per hr): 11.03 - 11.50
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 Mar 2016 To: 29 Mar 2016
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
The test substance was moistened with water.
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal skin
- % coverage: 10% of the body surface
- Type of wrap: The applied test substance was covered with a gauze patch, the latter being fixed with a gauze bandage wrapped around the animal and secured with tape.

REMOVAL OF TEST SUBSTANCE
- Washing: The test substance was wiped off with water.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied: 2000 mg/kg bw: males: 480 - 514 mg, females: 412 - 472 mg
- Constant volume used: no
- For solids, paste formed: The test substance was moistened with water.
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for mortality twice a day throughout the observation period. All animals were observed for clinical signs of toxicity at 30 min, 1 h, 2 h, and 4 h after treatment on the day of treatment and once daily thereafter for 14 days. The body weights of rats were individually recorded at one day prior to treatment (Day 0), on the day of treatment (Day 1), and at weekly intervals thereafter.
- Necropsy of survivors performed: yes
Statistics:
Body weight gain and group mean values were calculated over Day 0 body weights.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
no indication of skin irritation up to the relevant limit dose level
Mortality:
No mortality occurred during the study period.
Clinical signs:
No clinical signs of toxicity were observed up to the end of the 14-day observation period. No skin reaction was observed at the site of application in treated rats during this observation period.
Body weight:
Body weight gains of all animals were within the normal ranges in males and females during the whole study period.
Gross pathology:
Necropsy revealed no substance-related findings.

Table 1. Acute dermal toxicity

Dose

[mg/kg bw]

Mortality

Clinical signs

 

N*

N*

Males

2000

0/5

0/5

Females

2000

0/5

0/5

*N = number of animals/number of animals used

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Conclusions:
In the present acute dermal toxicity study in rats no mortality occured after single topical application of the limit dose level of 2000 mg/kg bw of the test substance for 24 h.
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Regulation (EC) No 1907/2006.

Additional information

Oral

The acute oral toxicity of the test substance was investigated in Wistar rats according to OECD test guideline (TG) 423 and GLP (M-580692-01-1, 2016). In accordance with the acute toxic class method, 2 groups of 3 female rats each received consecutively the test substance by oral gavage at a single dose of 2000 mg/kg bw. The test substance was suspended in water with 0.2% Tween 80. Following treatment, rats were observed for clinical signs of toxicity (once daily) and mortality (twice daily) for 14 days. Body weights were measured on a weekly basis. At study termination, all animals were subjected to gross necropsy. During the 14-day study period, neither mortality nor clinical signs of toxicity were observed. No effect on body weight/ body weight change was noted. At necropsy, no macroscopic alterations were found. Under the conditions of this study, the oral LD50 value for female rats was >2000 mg/kg bw.

Further, a second acute oral toxicity study is available in rats which was performed previously according to OECD TG 401 and GLP (M-192389-01-2, 1997b). In this study, 2 groups of 5 animals per sex and dose were administered the test substance in vehicle (0.5% methylcellulose in water) by gavage at doses of 5000 and 7080 mg/kg bw, respectively. The control group received vehicle only at the same dose volume as the treated groups. On Day 2, one male and two females treated with 5000 mg/kg bw died. These animals showed clinical signs of reduced motor activity, palpebral ptosis and hunched posture. On Day 3, a further female of the 7080 mg/kg bw dose group died without exhibiting clinical signs of toxicity. From this day on, no clinical signs of toxicity were observed up to the end of the 14-day observation period for both dose levels. No effects on body weights were observed and necropsy revealed no substance-related findings. Under the conditions of this study, the oral LD50 value for male and female rats was >7080 mg/kg bw.

In addition to the studies performed in rats, an acute toxicity study in rabbits was conducted similar to OECD TG 401 and according to GLP (M-416191-01-1, 2011). This study was not designed to derive a LD50 value of the test substance, but to establish an acute reference dose based on the investigation of total cytochrome P-450 levels, the activity of specific Phase I and II metabolic enzymes in liver, changes in thyroid hormones as well as morphological and histopathological alterations in liver and thyroid gland (see “Specific investigations: other studies”). In this study, 30 female animals (divided into two subgroups of 15 animals each) were treated with the test substance in 0.5% aqueous methylcellulose per oral gavage at dose levels of 0.85, 1.5 and 3.0 mg/kg bw. The control group was administered corresponding dose volumes of the vehicle only. Following an observation period of 24 h (group 1) or 14 days (group 2), no mortality and no clinical signs were noted at any dose level. The body weights were unaffected by treatment and necropsy revealed no substance-related findings at both time points. Due to the specific study design, no LD50 was derived from this study.

 

Inhalation

The acute inhalation toxicity of the test substance was studied in Wistar rats according to OECD TG 403 and GLP (M-610924-01-1, 2016). In a preliminary experiment, 1 male and 1 female animal were exposed to an atmosphere (dust) of the test substance at a concentration of 2.5 mg/L for 4 h. Following clinical signs of toxicity were observed in the female animal post exposure: laboured respiration (slight to severe), clonic convulsion and decreased activity (slight). The male animal shwoed the following symptoms post exposure: laboured respiration (slight to severe), noisy respiration (moderate to severe), incoordination (slight to moderate), decreased activity (slight to moderate), and irritability. The female animal died after the exposure on Day 0. The male animal survived and was symptom free from Day 7 onwards. Slight body weight loss (9.4%) was observed in the surviving male animal after the exposure, which was followed by normal body weight gain till study termination. Since severe effects and mortality were observed, the main study was performed at test concentrations of 2.5 and 1.25 mg/L (nominal). In the main study, 5 male and 5 female rats were exposed to an analytical atmosphere concentration of the test substance of 2.51 and 1.22 mg/L (dust) for 4 h using a nose only exposure system. The achieved MMAD ranged between 2.39 - 2.92 µm (GSD; 2.81 - 2.92). Following exposure, animals were observed for 14 days for clinical signs of toxicity and mortality. Body weights were measured on Day 0, 1, 3, 7, and 14. At study termination, all animals were subjected to gross necropsy.

At 2.51 mg/L, 2/5 male rats died during the exposure, 1 male and 2 females after the exposure on Day 0, 2 female animals were found dead on Day 1 and 1 female on Day 2 (total deaths: 3/5 males and 5/5 females). Animals exhibited following clinical signs of toxicity: laboured respiration (slight to severe), noisy respiration (slight), sneezing, decreased activity (slight to moderate), irritability and tonic convulsions. The 2 surviving male animals were symptom free from Day 5 till study termination.

At 1.22 mg/L, 1/5 females died after the exposure on Day 0. Clinical signs of toxicity were observed as laboured respiration (slight to severe), noisy respiration (slight), sneezing, decreased activity (severe), irritability and tremor (intermittent). The animals were symptom free from Day 4 onwards.

Slight to moderate body weight losses were observed in all animals following exposure. At necropsy, dark/red discoloration of lungs, and collapsed or enlarged lungs were observed.

Based on the results of this study, the LC50 is considered to be between 1.22 mg/L and 2.51 mg/L.

The following classification under Regulation (EC) No 1272/2008 (CLP) is warranted: Acute Tox. 4, Inhalation, H332

In a second acute inhalation toxicity study, the acute toxic potential of the test substance was studied in Sprague Dawley rats according to OECD TG 403 and GLP (M-192432-01-1, 1998). In a preliminary experiment, a single female rat was exposed to an atmosphere of the test substance at a concentration of 2.8 mg/L for 2 h. Since no severe effects were observed after exposure, the main study was performed at the limit test concentration of 5 mg/L (nominal). In the main study, 5 male and 5 female rats were exposed to an analytical atmosphere concentration of the test substance of 5.21 mg/L (5210 mg/m³; MMAD: 3.6 µm; GSD: 0.28) for 4 h using a nose only exposure system. Following exposure, animals were observed for 14 days for clinical signs of toxicity and mortality. Body weights were measured on Day -1, 0, 7, and 14. At study termination, all animals were subjected to gross necropsy. Clinical signs observed during the study included wet fur, hunched posture, piloerection, lethargy, ptosis, red/brown staining around the eyes and snout and on the head, and isolated incidents of decreased respiratory rate, ataxia, occasional body tremors and sneezing. During the 14-day observation period, animals recovered to appear normal within 1 or 2 days after exposure. No treatment-related abnormalities were observed at necropsy except for 4 treated males with dark foci on the lungs. Normal body weight gain was noted during the study apart from one female, which showed a slight loss in body weight during Week 1. Since no mortalities occurred during study, a LC50 value for male and female rats >5210 mg/m³ was derived.

Following a worst case assumption, the results of the most recently performed acute inhalation toxicity study are considered as sufficiently conclusive for classification as Acute Tox. 4, Inhalation, H332 according to Regulation (EC) No 1272/2008 (CLP).

 

Dermal

The acute dermal toxicity of the test substance was tested in accordance with OECD TG 402 and in compliance with GLP (M-580698-01-1, 2016). The study was performed as a limit test in Wistar rats (5 males and 5 females) at a dose level of 2000 mg/kg bw. The test substance (480 - 514 mg for males and 412 - 472 mg for females) was applied unchanged onto the clipped skin of the test animals for 24 h under semiocclusive conditions. After removal of the test substance, animals were observed for 14 days and sacrificed thereafter for gross pathological examinations. No treatment-related mortalities occurred and no signs of systemic toxicity were observed. The body weight gain in all animals was not affected by treatment. No test substance-related abnormalities were found at macroscopic post-mortem examination of the animals. Based on these results, the dermal LD50 value for male and female rats was > 2000 mg/kg bw.

These results were supported by a second acute dermal toxicity test which was performed previously also in accordance with OECD TG 402 and in compliance with GLP (M-192398-01-1, 1997a). Similarly, the study was performed as a limit test in Wistar rats (5 males and 5 females) at a dose level of 2000 mg/kg bw. The test substance (396 - 406 mg for males and 330 - 348 mg for females) was applied unchanged onto the shaved skin of the test animals for 24 h under semiocclusive conditions. After removal of the test substance, animals were observed for 14 days and sacrificed thereafter for gross pathological examinations. No treatment-related mortalities occurred and no signs of systemic toxicity were observed. The body weight gain in all animals was not affected by treatment. No test substance-related abnormalities were found at macroscopic post-mortem examination of the animals. Non-treatment related effects were observed in the lung (moderate haemorrhage) of 1/5 male and in the spleens (white, moderate to marked spots) of 2/5 female rats. Based on these results, the dermal LD50 value for male and female rats was >2000 mg/kg bw.

Justification for classification or non-classification

The available data on acute oral and dermal toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

The available data on acute inhalation toxicity of the test substance meet the criteria for classification as Acute Tox. 4, Inhalation (H332) according to Regulation (EC) 1272/2008.