Registration Dossier

Administrative data

Description of key information

An acute oral toxicity study (toxic class method)  and an acute inhalation study were conducted.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and OECD/EC guidelines
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 8-11 Weeks
- Weight at study initiation: 155-187g
- Fasting period before study: 16-19 hours
- Housing: IVC cages, type III H, polysulphone; Altromin saw fibre bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degrees C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): 10x /hr
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- water
- Amount of vehicle (if gavage): 10mL/kg bodyweight

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg
Doses:
2000, 300, 50 mg/kg
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 weighing: day 1 (prior to dosing) and days 8 & 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross necropsy
Sex:
female
Dose descriptor:
LD50
Effect level:
200 mg/kg bw
Mortality:
100% mortality was observed at 2000 and 300 mg/kg 1-2 minutes post dosing, 0% mortality at 50 mg/kg
Clinical signs:
clonic convulsions and opisthotonos were associated with all mortalities along with lateral recumbency, kyphosis and half eyelid closure in the high dose group.

Clonic convulsions, opisthotonos, prone position, piloerection were seen at up to 30 minutes post-dose in 2 of the 6 animals treated at 50 mg/kg, with much reduced clinical signs (such as slight piloerection, prone position) seen in the other 4 animals. nosigns of toxicity were seen from 1d until the end of the observation period in any of the animals.
Body weight:
Body weight gain was normal for the surviving animals in this study.
Gross pathology:
No significant findings for animals dosed at 50 mg/kg.
Animals dosed at 2000 mg/kg had residual test item in the stomach.
Animals dosed at 300 mg/kg had stomachs filled with liquid and bloody spots in the brain.
Interpretation of results:
toxic
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The median lethal dose after single oral administration to female rats observed over 14 days was:
LD50 cut off (rat) = 200 mg/kg body weight.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
200 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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD guidelines and GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 9 - 12 weeks old
- Weight at study initiation: Bodyweight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: No
- Housing: Group housing of 5 animals per cage in labelled makrolon cages containing sawdust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet except during exposure.
- Water (e.g. ad libitum): Free access to tap water except during exposure.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour light, 12 hour dark cycle.

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The design of the chamber is based on the flow past nose-only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983).
- Exposure chamber volume: Approximately 150 mL
- Method of holding animals in test chamber: Animals were placed in restraining tubes.
- Source and rate of air: Pressurised air at a rate of 22 L/min for the initial exposure, and 17 L/min for the second exposure.
- Method of conditioning air: None, pressurised air used to supply an adequate oxygen supply.
- System of generating particulates/aerosols: For the initial exposure (ca. 1 mg.L), the test substance was administered in a stream
- Method of particle size determination: Samples collected with an 8 stage Marple personal cascade impactor containing fibre glass filters, and a fibre glass back up filter. Amounts of test substance collected were measure gravimetrically and the MMAD and GSD determined.
- Treatment of exhaust air: Air passed through a filter before being released into the exhaust of the fume hood.
- Temperature, humidity, pressure in air chamber: For ca. 1 mg/L the temperature ranged between 21.6 and 22.4°C with humidity between 11 and 21% and at ca. 7 mg/L temperature was 21°C and humidity was at 5%. These conditions were considered appropriate for the relatively short exposure duration.

TEST ATMOSPHERE
- Brief description of analytical method used: A total of 13 and 4 representative samples were taken for determination of the actual concentration during exposure at ca. 1 and ca. 7 mg/L, respectively. Samples were drawn from the test atmosphere through a tube mounted in a free animal port and through a glass fibre filter. The collected amount of test material was measured gravimetrically, and measured by means of a dry gas meter. The time-weighted means and standard deviations were calculated.


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: At ca. 1 mg/L the MMAD and GSD were determined twice. The MMAD was 3.9 μm (gsd 1.6) and 4.5 μm (gsd 1.6). The MMAD values are at teh upper end of 1 - 4 μm. As one measurement was above the upper limit, and based on the effects on the animals, it can be assumed that sufficient deposition in the lower respiratory tract occured. At ca. 7 mg/L, the MMAD and gsd were not determined during the exposure, since the exposure was terminated after 32 minutes. Since the test substance was grinded in the same way as for the ca. 1 mg/L exposure and taken into account the effects on the animals, sufficient lung deposition can be expected. There was no evidence of test substance deposits in the upper air ways.


CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Based on effects observed in the acute oral study, a starting concentration of ca. 1 mg/L was considered appropriate.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
ca. 4 h
Remarks on duration:
At 1 mg/L, at ca. 7 mg/L exposure duration was 32 minutes due to observed toxicity
Concentrations:
For the 1 mg/L group, the time-weighted mean actual concentration was 1.1 +/-0.04 mg/L. This was considered sufficiently stable to represent a 4 hour exposure to 1 mg/L.

For the second exposure targeted at 5 mg/L, the actual time-weighted mean concentration was 6.8 +/- 0.88 mg/L. Due to the mortality and signs of the animals already observed after 32 minutes, the exposure was terminated after 32 minutes. The concentration was measured four times during this exposure and showed a variation between 2 and 12 mg/L. Based on the early manifestation of the mortality and clinical signs, within 32 minutes after exposure and the variability in concentration range of 2 and 12 mg/L, it was not considered possible to determine whether mortality was due to a higher than expected initial exposure or whether the 4-hour LC50 would fall above or below 5 mg/L. There was no evidence of test substance overload of the respiratory tract.
No. of animals per sex per dose:
5 males and 5 females per dose.
Control animals:
no
Details on study design:
- Duration of observation period following administration: Up to Day 15 for surviving animals.
- Frequency of observations and weighing: Clinical signs were observed three times during exposure, shortly after exposure and then once daily until day 15. Bodyweights were recorded pre-exposure, and on days 2, 4, 8 and 15 and at death.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, bodyweights and macroscopic pathology.
Statistics:
No statistical analysis was performed, as it not a requirement on studies on this type.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 mg/L air (nominal)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
At ca. 1 mg/L no mortality occured.

At ca. 7 mg/L, three males and two females were found dead and one female was sacrificed for ethical reasons between 34 and 44 minutes after the start of exposure. One males was sacrificed for ethical reasons on Day 2. No further mortality occured for the remaining male and two females.
Clinical signs:
At ca. 1 mg/L, during exposure, no abnormalities were seen.
After exposure, lethargy (1 male), flat posture (1 male), hunched posture (all animals), laboured respiration (2/5 males, 1/5 females), gasping (1 male), chromodacryorrhoea (1 male) and/or yellow urine (all animals) were seen for the animals between Days 1 and 4. One male showed lethargy and hunched posture between Days 7 and 13. One female showed rales on Days 14 and 15.

At ca. 7 mg/L, tonic-clonic spasms were seen for the animals during exposure. Immediately after termination of the exposure, tonic-clonic spasms, tremors, hunched posture, abnormal gait, laboured respiration and flat posture were seen for the animals. From 32 minutes after termination of the exposure onwards, lethargy (2/5 males and females), tremor (2/5 males and females), flat posture (2/5 males), hunched posture (2/5 males and females), laboured respiration (2/5 males and females) and rales (2/5 males and females) were seen for the animals. The surviving animals had recovered from the signs between Days 6 and 9.
Body weight:
At ca. 1 mg/L, overall body weight gain in four males and all females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity. One male showed body weight loss up to Day 8 and regained weight during the second week post-exposure.

At ca. 7 mg/L, overall body weight gain for the two surviving females was within the range expected for rats of this strain and age used in this type of study and were therefore considered not indicative of toxicity. Body weight loss was noted in the surviving male during the first week post-exposure and the male regained weight during the second week post-exposure.
Gross pathology:
At ca. 1 mg/L, no abnormalities were found at macroscopic post mortem examination of the animals.

At ca. 7 mg/L, macroscopic examination for the animals that died or were sacrificed for ethical reasons during the study revealed several dark red foci for the lungs (one male), pelvic dilation of the kidneys (three males), dark red discolouration of the mandibular lymph nodes (one male and one female), enlarged thymus (one male) and diaphragmatic hernia of the right medial lobe of the liver (one female). No abnormalities were found for the three surviving animals.

Beginning autolysis was found for one animal found dead which was considered not related to treatment.
Interpretation of results:
harmful
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The LC50 (4 hour) is normally ranked within the following ranges (0-0.05, 0.05-0.5, 0.5-1, 1-5 or >5 mg/L). However, given the actual concentrations obtained at target concentrations of 1 and 5 mg/L (1.1 and 6.8 mg/L) it was considered appropriate to rank the LC50 (4 hour) as > 1 mg/L.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
1 000 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The median lethal dose after single oral administration to female rats observed over 14 days was:

LD50 cut off (rat) = 200 mg/kg body weight.

100% mortality was observed at 2000 and 300 mg/kg, 0% mortality at 50 mg/kg

The median lethal dose after a maximum 4 hour exposure to male and female rats was:

LC50 (4 hour) = > 1 mg/L (> 1000 mg/m3).


Justification for selection of acute toxicity – oral endpoint
Key study via the oral route conducted to OECD guidelines and GLP.

Justification for selection of acute toxicity – inhalation endpoint
Key study via the inhalation route conducted to OECD guidelines and GLP.

Justification for classification or non-classification

Based on the outcome of the acute oral (toxic class) study, the substance should be classified for acute toxicity (category 3) according to the CLP Regulation (EC) 1272/2008 as amended.

Based on the outcome of the acute inhalation study, the substance should be classified for acute toxicity (category 4) according to the CLP regulation (EC) 1272/2008, as amended.