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Administrative data

Description of key information

Acute Oral Rat

Under the conditions of this study, the acute oral LD50 was determined to be 79.84 mg/kg and 131.98 mg/kg in male and female rats respectively. 

Acute Oral Mouse

Under the conditions of this test, the acute oral LD50 of the test material in the male mouse was determined to be 155 mg/kg. In the female mouse, the oral LD50 was determined to be 214 mg/kg. In the sexes combined, the oral LD50 was determined to be 179 mg/kg .

Acute Inhalation

Under the conditions of this study, the acute inhalation LC50 was 691 ppm in both male and female rats.

Acute Dermal

Under the conditions of this study the percutaneous LD50 was found to be in excess of 2000 mg/kg for both male and female rabbits.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 April 2000 to 6 June 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 59 NouSan No. 4200
Version / remarks:
1985
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: range-finder: males and females-11 weeks, main study: males-10 to 13 weeks and females-10 to 12 weeks
- Weight at study initiation: range-finder: males-306 to 358 g and females- 205 to 216 g, main study: males-283 to 399 g and females- 208 to 270 g
- Fasting period before study: overnight before dosing
- Housing: The animals were housed individually in suspended stainless steel cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 22 °C
- Humidity: 37 to 59 %
- Air changes: room ventilation was set to produce 10-15 air changes/hour.
- Photoperiod: Light timers were set to maintain a 12-hour light/12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSAGE PREPARATION:
- The test material was administered as received or mixed with corn oil to produce a 10 % w/v concentration and dispensed fresh on the day of dosing. The preparations were stirred continuously during dosing.
- The density of the test material was determined twice to confirm the volatile nature of the test material. The average of the two density measurements resulted in 2.22 g/mL. The density of the test material (bulk) was determined to be 2.22 g/mL.
- For the range finding study the test material was dosed as supplied for all concentrations. The maximum volume applied was 2.25 mL/kg
- For the main LC50 study at 50 and 75 mg/kg the test material was dosed from the 10 % w/v dilution prepared in corn oil. For all other dose groups the test material was dosed as supplied (100 %).

- Rationale for the selection of the starting dose: The range-finding data indicated that the test material produced mortality at the 500 mg/kg level in males and in females. Delayed mortality (day 5) was observed at the lowest dose level tested (100 mg/kg) in males, but no mortality occurred in the females.
Doses:
Range finding study: 100, 500, 1250, 2500 and 5000 mg/kg bw
Main LC50 study: 50, 75, 85, 100, 150, 250, 350 mg/kg bw
No. of animals per sex per dose:
- Range finding study: 1 animal per sex per dose
- Main LC50 study:
50 mg/kg: 5 animals per sex per dose
75 mg/kg: 5 males
85 mg/kg: 5 males
100 mg/kg: 5 animals per sex per dose
150 mg/kg: 5 females
250 mg/kg: 5 animals per sex per dose
350 mg/kg: 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed for clinical abnormalities a minimum of two times on study day 0 (post-dose) and daily thereafter (days 1-14). A general
health/mortality check was performed twice daily (in the morning and in the afternoon).
- Individual body weights were obtained for the study animals prior to fasting (day -1), prior to dosing on day 0 and for all surviving animals on days 7 and 14.
- Necropsy of survivors performed: yes, all study animals which died spontaneously during the study or were euthanised by carbon dioxide inhalation at study termination (day 14) were necropsied. Body cavities (cranial, thoracic, abdominal and pelvic) were opened and examined. All gross lesions were collected and retained in 10 % neutral buffered formalin for possible future histological evaluation.
Statistics:
The LD50 and 95 % confidence intervals were calculated separately for each sex using a computer adaptation of the method of Litchfield and Wilcoxon.
Preliminary study:
The range-finding data indicated that the test material produced mortality at the 500 mg/kg level in males and in females. Delayed mortality (day 5) was observed at the lowest dose level tested (100 mg/kg) in males, but no mortality occurred in the females.
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
79.84 mg/kg bw
Based on:
test mat.
95% CL:
>= 77.25 - <= 82.55
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
131.98 mg/kg bw
Based on:
test mat.
95% CL:
>= 95.35 - <= 182.7
Mortality:
- All male rats died at the 85 mg/kg dose. No males died at the 75 mg/kg dose.
- For females, all rats died at the 350 mg/kg high dose but none at the 50 mg/kg low dose. Partial deaths were observed at the intermediate doses.
- All mortality occurred by study day 2.
- A summary is shown in Table 1.
Clinical signs:
- The most notable clinical abnormalities observed during the study included breathing abnormalities, prostration, tremors, soft stools/few faeces, faecal stain, dilated pupils, skin pale in colour, ocular discharge, salivation, decreased activity, wobbly gait, eyelids partially closed and dark material around the facial area.
- Some clinical observations were seen in all dose groups.
Body weight:
- Body weight gain/maintenance was noted for all surviving animals during the test period.
Gross pathology:
- The most notable gross internal findings were observed in the animals that died and included abnormal content of the digestive tract, foci/thickening of the stomach, mottled and/or dark red lung lobes, foci on the thymus and blackish-purple lobes of the liver. No significant gross internal findings were observed at necropsy on study day 14 .

Table 1: Summary of mortalities

Dose level (mg/kg)

Sex

No. of animals

Study day

Mortality

0

1

2

3 to 14

50

Male

5

0

0

0

0

0/5

75

5

0

0

0

0

0/5

85

5

4

1

-

-

5/5

100

5

4

0

1

-

5/5

250

5

5

-

-

-

5/5

50

Female

5

0

0

0

0

0/5

100

5

1

0

0

0

1/5

150

5

3

0

1

0

4/5

250

5

4

0

0

0

4/5

350

5

5

-

-

-

5/5

Interpretation of results:
other: Category 3 according to EU criteria.
Conclusions:
Under the conditions of this study, the acute oral LD50 was determined to be 79.84 mg/kg and 131.98 mg/kg in male and female rats respectively. 
Executive summary:

The acute oral toxicity of the test material was determined in accordance with the standardised guidelines EPA OPPTS 870.1100 and JMAFF 59 NohSan No. 3850, under GLP conditions.

The single-dose oral toxicity of the test material was evaluated in Sprague-Dawley rats. An LD50 study was performed in which 5 groups of five male and five female rats received a single oral administration of the test material at graded dosage levels. Following dosing, the LD50 study rats were observed daily and weighed weekly. A gross necropsy examination was performed on all LD50 study animals at the time of death or scheduled euthanasia (day 14).

All male rats died at the 85 mg/kg dose. No males died at the 75 mg/kg dose. For females, all rats died at the 350 mg/kg high dose but none at the 50 mg/kg low dose. Partial deaths were observed at the intermediate doses. All mortality occurred by study day 2. The most notable clinical abnormalities observed during the study included breathing abnormalities, prostration, tremors, soft stools/few faeces, faecal stain, dilated pupils, skin pale in colour, ocular discharge, salivation, wobbly gait, decreased activity, eyelids partially closed, and dark material around the facial area. Body weight gain/maintenance was noted for all surviving animals during the test period. The most notable gross internal findings were observed in the animals that died and included abnormal content of the digestive tract, foci/thickening of the stomach, mottled and/or dark red lung lobes, foci on the thymus and blackish-purple lobes of the liver. No significant gross internal findings were observed at necropsy on study day 14.

Under the conditions of this study, the acute oral LD50 was determined to be 79.84 mg/kg and 131.98 mg/kg in male and female rats respectively. 

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2000 to 05 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: For the range-finding study, the males and females were approximately 8 weeks of age. For the LD50 study, the males were approximately 9-11 weeks of age and females were approximately 9-12 weeks of age.
- Weight at study initiation: For the range-finding study, the males weighed 30-35 g and the females weighed 25-28 g prior to fasting. For the LD50 study, the males weighed 28-38 g prior to fasting and the females weighed 24-31 g prior to fasting
- Fasting period before study: yes
- Housing: The animals were housed individually in suspended stainless steel cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17- 21 °C (63-69 °F)
- Humidity: 42-76 %
- Air changes: room ventilation was set to produce 10-15 air changes/hour
- Photoperiod: Light timers were set to maintain a 12-hour light/ 12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
DOSAGE PREPARATION:
The test material was mixed with corn oil to produce the 10 % w/v concentration for dose administration. The test material was prepared/dispensed fresh on the day of dosing and stirred continuously during dosing. The density of the test material was determined twice to confirm the volatile nature of the test material. The average of the two density measurements resulted in 2.22 g/mL. Note: In order to maintain an accurate dose volume during the study (due to the low dose levels and the small size of the animals) without having to vary both the dose volume and the concentration, the test material was dosed at the 10 % w/v concentration.
Doses:
- Range-finding study: 50, 100, 250, 500 and 1000 mg/kg
- LD50 study: 100, 175, 200, 225 and 250 mg/kg
No. of animals per sex per dose:
1 animal per sex per dose in the range-finding study and 5 animals per sex per dose in the LD50 study.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
The animals were observed for clinical abnormalities a minimum of two times on day 0 (post-dose) and daily thereafter (days 1-14). A general health/mortality check was performed twice daily (in the morning and in the afternoon).
- Individual body weights were obtained for the animals prior to fasting (day 0), prior to dosing on day 0 and for all surviving animals on days 7 and 14.
- Necropsy of survivors performed: yes, all animals that died spontaneously during the study or were euthanised by carbon dioxide inhalation at study termination (day 14) were necropsied. Body cavities (cranial, thoracic, abdominal and pelvic) were opened and examined. All gross lesions were collected and retained in 10 % neutral buffered formalin for
possible future histological evaluation
Statistics:
- The LD50 and 95 % confidence interval were calculated separately for males, females and the combined sexes using a computer adaptation of the method of Litchfield and Wilcoxon.
- Body weight means and standard deviations were calculated separately for males and females for each level administered .
Preliminary study:
The range-finding data indicated that the test material produced mortality at the 250 mg/kg level in males and in females. No mortality was observed at the next
lower dose level (100 mg/kg).
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
214.1 mg/kg bw
Based on:
test mat.
95% CL:
>= 200 - <= 229.1
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
155 mg/kg bw
Based on:
test mat.
95% CL:
>= 130.1 - <= 184.7
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
179.2 mg/kg bw
Based on:
test mat.
95% CL:
>= 162.2 - <= 197.9
Mortality:
All mortalities in the study occurred by day 3, mortalities are summarised in Table 1.
Clinical signs:
The most notable clinical abnormalities observed during the study included soft stool(s), urine/faecal stain, decreased/no defecation, decreased food consumption, dilated pupil(s), eyelids partially closed, piloerection, rough haircoat, prostration, apparent hypothermia, decreased activity, breathing abnormalities, salivation, skin blue in colour - entire body, hunched posture and wobbly gait.
Body weight:
In the 100 mg/kg dose group, body weight loss was noted for one male and one female during the day 0-7 body weight interval and one male during the day 7-14 body weight interval. Two male and two female mice in the 175 mg/kg dose group lost body weight during the day 0-7 body weight interval. Two 200 mg/kg females lost body weight during the day 0-7 body weight interval and one female lost body weight during the day 7-14 body weight interval. In the 225 mg/kg dose group, two females lost body weight during the day 0-7 body weight interval. Body weight gain or maintenance was noted for the remaining surviving animals during the test period.
Gross pathology:
- Significant gross internal findings observed in the animals found dead were abnormal content of the digestive tract, dilated pelvis of the kidneys, reddened glandular mucosa of the stomach and red fluid in the thoracic cavity.
- At scheduled euthanasia (day 14), the significant gross internal findings which were apparently test material related based on the number of animals with these findings included thickened areas of the stomach associated with adhesion involving the body wall and stomach serosa. Three incidences of periovarian cysts were observed but were not considered test material related or significant since this is commonly found in animals of this strain.

Table 1: Summary of mortality

Dose Level

Male/ Female

No. of animals

Study day

Mortality

0

1

2

3

4-14

100

Male

5

0

0

0

0

0

0

175

5

0

0

1

1

0

2

200

5

0

4

1

0

0

5

225

5

0

5

0

0

0

5

250

5

0

4

1

0

0

5

100

Female

5

0

0

0

0

0

0

175

5

0

0

0

0

0

0

200

5

0

1

0

0

0

1

225

5

0

3

0

0

0

3

250

5

0

4

1

0

0

5

Interpretation of results:
other: Category 3 according to EU criteria.
Conclusions:
Under the conditions of this test, the acute oral LD50 of the test material in the male mouse was determined to be 155 mg/kg. In the female mouse, the oral LD50 was determined to be 214 mg/kg. In the sexes combined, the oral LD50 was determined to be 179 mg/kg.
Executive summary:

The acute oral toxicity of the test material was determined in accordance with the standardised guideline OPPTS870.1100, under GLP conditions.

The single-dose oral toxicity of the test material was evaluated in CD-1 mice. The study was initiated with a range-finding test at levels ranging from 50 to 1000 mg/kg body weight. Following the range-finding test, an LD50 study was performed in which groups of five male and five female mice received a single oral administration of the test material at graded dosage levels (five males and five females per level). Following dosing, the LD50 study mice were observed daily and weighed weekly. A gross necropsy examination was performed on all animals at the time of death or scheduled euthanasia (day 14).

All mortality occurred by study day 3. The most notable clinical abnormalities observed during the study included soft stools, slight to severe urine stain, slight to moderate faecal stain, decreased/no defecation, decreased food consumption, salivation, dilated pupil(s), eyelids partially closed, piloerection, rough haircoat, prostration, apparent hypothermia, decreased activity, breathing abnormalities, skin blue in colour - the entire body, hunched posture and wobbly gait. In the 100 mg/kg dose group, body weight loss was noted for one male and one female during the day 0-7 body weight interval and one male during the day 7-14 body weight interval. Two male and two female mice in the 175 mg/kg dose group lost body weight during the day 0-7 body weight interval. Two 200 mg/kg females lost body weight during the day 0-7 body weight interval and one female lost body weight during the day 7-14 body weight interval. In the 225 mg/kg dose group, two females lost

body weight during the day 0-7 body weight interval. Body weight gain or maintenance was noted for the remaining surviving animals during the test period. Significant gross internal findings observed in the animals found dead were abnormal content of the digestive tract, dilated pelvis of the kidneys, reddened glandular mucosa of the stomach and red fluid in the thoracic cavity. At scheduled euthanasia, the significant gross internal findings were thickened areas of the stomach associated with adhesion involving the body wall and stomach serosa.

Under the conditions of this test, the acute oral LD50 of the test material in the male mouse was determined to be 155 mg/kg. In the female mouse, the oral LD50 was determined to be 214 mg/kg. In the sexes combined, the oral LD50 was determined to be 179 mg/kg .

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
79.84 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:
12 December 2000 to 04 January 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 12 NouSan No. 8147
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 to 12 weeks of age
- Weight at study initiation: 222 to 292 g
- Fasting period before study: Food and water were witheld during the exposure
- Housing: Individual suspended wire-mesh cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 22 °C
- Humidity: 54 to 61 %
- Photoperiod: 12 hours light/ 12 hours dark

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass and stainless steel whole body exposure chamber
- Exposure chamber volume: 130 L
- Method of holding animals in test chamber: The animals were caged individually during exposure
- Temperature, humidity, pressure in air chamber: Exposure atmosphere conditions were recorded four times during the exposure.
- Vapour atmospheres of the test material were generated using the following system: The test material was held in a glass gas washing bottle. Compressed air (generation air) was delivered to the washing bottle to create a vapour of the test material. The resulting vapour was carried to the chamber with the appropriate flow of dilution air. Humidified purge air was also supplied to the chamber to help maintain the protocol-specified humidity range. Exhaust vapour from the chamber was filtered (charcoal) prior to entering the facility exhaust system.
- Generation airflow; 581 ppm: 41 to 49 mL/min, 710 ppm: 51 to 60 mL/min, 797 ppm: 65 to 68 mL/min and 1189 ppm: 110 to 128 mL/min.
- Dilution airflow: 11 L/min for all test concentrations
- Purge airflow rate: 11 L/min for all test concentrations

TEST ATMOSPHERE
- The nominal exposure concentration was calculated as the total amount of test material used during each exposure divided by the total volume of air that passed through the system during that exposure.
- Actual exposure concentrations were measured using gas chromatography. Samples of the exposure atmospheres were manually collected from the chamber using a sampling valve and sample loop. The gas chromatograph (GC) conditions were:
- Instrument: Hewlett Packard 5890A with a 3396A integrator
- Detector: Flame ionisation
- Column: DB-W AX/0.25 µm 30 m x 0.25 mm
- Gases: Carrier-Helium: 53 psig (8 L/min), Fuel hydrogen: 18 psig (31 mL/min) and Air: 35 psig (300 mL/min) .
- Temperatures (°C): Detector: 250, Column: 45 and Injector: 250
- Injection volume: 0.25 mL
- Retention time: Approximately 1.1 minutes
- Samples taken from breathing zone: yes/no
- The GC was standardised using known concentrations of the test material prepared in Tedlar® gas bags. The bags were prepared by injecting known volumes of test material into a glass vaporisation bulb. A continuous flow of air through the bulb carried the vaporised test material to a 40 L bag. The volume of air was measured by a dry test meter. Concentrations of the gas phase standards were calculated as follows:
Concentration (ppm) = (VOL x R x T x D x 10^-3 x 10^6 ) / (L x GMA x P)
Where VOL = volume of the test article (µL), R = universal gas constant (62.36 L mmHg/mol K), T = nominal laboratory temperature (294 K), D = density of test article (2.28), L = volume of air used to make bag (32 L), GMW = molecular weight of test material (141.94), P = laboratory barometric pressure (730 mmHg), 10^-3 = µL to mL conversion, 10^6 = ppm conversion
- Standards were prepared that bracketed the target concentration for each exposure. Each standard bag was prepared and analysed on the GC. An instrument response for each standard was recorded to produce a calibration curve. On the day of exposure, one standard was analysed to confirm GC calibration. Then for each exposure, the instrument response was converted to concentration from the calibration curve for each sample.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
581, 710, 797, 1189 ppm
Duration of exposure:
ca. 4 h
Concentrations:
Target concentrations of 600, 700, 800 and 1200 ppm (concentrations confirmed at 581, 710, 797 and 1189 ppm respectively)
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Each rat was observed immediately following exposure on day 0 and once daily thereafter for 14 days. Due to the limited visibility of the animals in the exposure chamber, observations during exposure were limited to an evaluation for mortality.
- Body weights were obtained prior to exposure on day 0 and on post-exposure days 3, 7 and 14. A final body weight was recorded for all animals found dead.
- Necropsy of survivors performed: yes, all animals found dead or euthanised after the 14-day observation period underwent a gross necropsy. Animals at the scheduled necropsy were euthanised by an intravenous injection of sodium pentobarbital. The major organ systems of the cranial, thoracic and abdominal cavities were examined for all animals.
Statistics:
The data did not warrant statistical analysis, however the LD50 and 95 % confidence intervals were calculated separately for each sex using a computer adaptation of the method of Litchfield and Wilcoxon.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
ca. 691 ppm
Based on:
test mat.
Exp. duration:
4 h
Mortality:
- For the 710 ppm group, one male died during exposure, one male and one female died following exposure on day 0 and two males and three females died on day 1. For the 797 ppm group, one male died following exposure on day 0, three males and three females died on day 1 and one female died on day 2. Four males and five females in the 1189 ppm group died during exposure and the one remaining male in the 1189 ppm group died within one hour of the end of the exposure on day 0.
- Mortalities can be seen in Table 1.
Clinical signs:
other: - Clinical signs can be seen in Table 2 and Table 3. - All animals in the 581 ppm group were considered normal by day 7. The surviving animals in the 710 and 797 ppm groups were considered normal by day 4.
Body weight:
- All animals in the 581 ppm group lost weight (6 to 22 g) from days 0 to 3. All animals in the 581 ppm group surpassed their initial day 0 body weight by day 7.
- In the 710 ppm group, body weight losses of 63 and 31 grams were noted from days 0 to 3 for the surviving male and female, respectively. Both of these animals surpassed their initial body weight by day 14.
- Similarly, the surviving male and female in the 797 ppm group lost 49 and 18 grams, respectively, from days 0 to 3 and surpassed their initial weight by day 14.
Gross pathology:
- A distended, gas-filled stomach was noted macroscopically for eight (four males, four females) of the 10 animals in the 1189 ppm group that died during exposure.
- For the animals in the 797 ppm group that died early, a distended intestine (parts or entire length) was observed for three males and one female, dark red adrenal glands and a haemorrhagic thymus gland were each observed for two females.
- Dark red lungs were observed for two males and a reddened pituitary gland was observed for one male in the 710 ppm group that died early. In addition, a distended, gas-filled stomach was observed for one male and a reddened glandular portion of the stomach was observed for two females in the 710 ppm group that died.
- There were no other toxicologically significant internal findings in the animals found dead.
- There were no internal macroscopic findings at the scheduled necropsies .
Other findings:
- At the nominal concentrations of 1046, 1369, 1542 and 2388 ppm the actual concentrations were 581, 710, 797 and 1189 ppm.

Table 1: Mortalities

Concentration (ppm)

Number dead/Number treated

Males

Females

Males and Females combined

581

0/5

0/5

0/10

710

4/5

4/5

8/10

797

4/5

4/5

8/10

1189

5/5

5/5

10/10

Table 2: Clinical signs observed immediately after exposure

Concentration (ppm)

Clinical sign

No. of animals

Males

Females

581

Hypoactivity

5

5

Clear material around the mouth, nose and/or eyes

5

5

Clear nasal discharge

4

4

Rales

4

3

Laboured respiration

5

1

Gasping

3

1

Red material around the nose

1

0

Ataxia

1

0

710

Hypoactivity

4

5

Clear material around the mouth, nose and/or eyes

4

5

Laboured respiration

3

3

Gasping

1

1

797

Hypoactivity

5

4

Clear material around the mouth, nose and/or eyes

5

5

Laboured respiration

4

3

1189

Hypoactivity

1

0

Clear material around the mouth, nose and/or eyes

1

0

Laboured respiration

1

0

Table 3:Clinical signs observed during the 14 day observation period

Concentration (ppm)

Clinical sign

No. of animals

Males

Females

581

Hypoactivity

5

5

Red material on various body surfaces

3

3

Rales

2

0

Decreased defecation/ urination

0

1

710

Hypoactivity

1

1

Rales

1

0

Laboured respiration

1

1

Gasping

1

0

Red material on various body surfaces

1

1

Decreased defecation/ urination

1

1

Mucoid faeces

0

1

Unkempt appearance

1

0

797

Hypoactivity

1

2

Red material on various body surfaces

1

2

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Under the conditions of this study, the acute inhalation LC50 was 691 ppm in both male and female rats.
Executive summary:

The acute inhalation toxicity of the test material was investigated in accordance with the standardised guidelines EPA OPPTS 870.1300 and JMAFF 8147 under GLP conditions.

The acute inhalation toxicity was evaluated in a four hour, single-exposure study in rats. The test material was administered to four groups of five male and five female albino rats each via whole-body exposure as a vapour atmosphere at concentrations of 581, 710, 797 and 1189 ppm. Following each exposure, all surviving animals were maintained for a 14-day observation period. Parameters evaluated were mortality, clinical observations, body weights and gross necropsy.

Eight animals in each of the 710 and 797 ppm groups and all 10 animals in the 1189 ppm group died within two days of exposure as a result of test article administration. There were no deaths in the 581 ppm group.

Ataxia, clear nasal discharge, gasping, hypoactivity, laboured respiration, rales and red material around the nose were observed immediately following exposure in the 581 ppm group. Gasping, hypoactivity and/or laboured respiration were observed for the surviving animals in 710, 797 and 1189 ppm groups immediately following exposure. During the 14-day observation period, decreased defecation/urination, hypoactivity, red material on various body surfaces and rales were noted in the 581 ppm group. In addition to these clinical findings, gasping, mucoid faeces, laboured respiration and unkempt appearance were also observed for the surviving animals in the 710 ppm group. For the surviving animals in the 797 ppm group, hypoactivity and red material on various body surfaces were noted. All surviving animals were considered clinically normal by day 4 for the 710 and 797 ppm groups and by day 7 for the 581 ppm group.

Body weight losses were observed for all animals in the 581, 710 and 797 ppm groups from days 0 to 3. All surviving animals surpassed their initial day 0 body weight by day 14.

Macroscopic findings were limited to the animals that died early. In the 1189 ppm group, a distended, gas-filled stomach was noted for eight animals. For animals found dead in the 797 ppm group, a distended intestine (parts or entire length) was observed for three males and one female and dark red adrenal glands and a haemorrhagic thymus gland were each observed for two females. Dark red lungs were observed for two males and a reddened pituitary gland was observed for one male in the 710 ppm group. In addition, a distended, gas-filled stomach was observed for one male and a reddened glandular portion of the stomach was observed for two females in the 710 ppm group.

Under the conditions of this study, the acute inhalation LC50 was 691 ppm in both male and female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
4 011 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:
04 May 2000 to 24 May 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 59 NouSan No. 4200
Version / remarks:
1985
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 14-16 weeks of age and females: 11-18 weeks
- Weight at study initiation: males 3.0 to 3.3 kg and females: 2.4 to 3. 7 kg
- Fasting period before study: no
- Housing: The animals were housed individually in suspended stainless steel cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 22 °C
- Humidity: 46 to 82 %
- Air changes: 10 to 15 air changes/hour
- Photoperiod: Light timers were set to maintain a 12-hour light/12-hour dark cycle
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: The dorsal trunk area. The region included the scapula (shoulder) to the wing of the ilium (hipbone) and half way down the flank on each side of the animal.
- % coverage: approximately 10 %
- Type of wrap if used: a 4-ply porous gauze dressing backed with a plastic wrap. Removal and ingestion of the test material was prevented by placing an elastic wrap over the trunk and test area. The elastic wrap was further secured with adhesive tape around the trunk at the cranial and caudal ends.

REMOVAL OF TEST SUBSTANCE
- Washing: Residual test material was removed using gauze moistened with deionised water followed by dry gauze.
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied: 500 mg/kg dose group: 0.23 mL/kg and 2000 mg/kg dose group: 0.90 mL/kg
- Concentration: The test material was administered as received. The density of the test material was determined twice to confirm the volatile nature of the test material. The average of the two density measurements resulted in 2.22 g/mL.

- After dosing, collars were placed on the animals and remained in place until removal on study day 3 for the 500 mg/kg dose level and on study day 14 for the 2000 mg/kg dose level.
Duration of exposure:
24 hrs
Doses:
500 and 2000 mg/kg
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- The test animals were examined for erythema and oedema and the responses scored following patch removal on study day 1 and daily thereafter (days 2 to 14) according to the Macroscopic Dermal Grading System which is based on Draize. The dermal test sites were re-clipped as necessary to allow clear visualisation of the skin.
- The test animals were observed for clinical abnormalities two times on study day 0 (post-dose) and daily thereafter (days 1 to 14). A mortality check was performed twice daily, in the morning and afternoon.
- Individual body weights were obtained for the test animals prior to dosing on day 0 and on days 7 and 14.
- Necropsy of survivors performed: yes, all animals were euthanised by an intravenous injection of sodium pentobarbital at study termination (day 14) and were necropsied. Body cavities (cranial, thoracic, abdominal and pelvic) were opened and examined. Treated skins samples were retained for one male and one female for possible future histopathological examination.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred during this study.
Clinical signs:
- Clinical abnormalities observed during the study included decreased defecation, soft stools, faeces small in size, decreased food consumption, breathing abnormalities and dark material around the facial area.
- Dermal irritation was noted at the site of test material application for every animal. The dermal irritation for the 2000 mg/kg dose level was severe and included apparent haemorrhaging at the test site. The severity of findings at the 2000 mg/kg dose level precluded dosing higher levels
Body weight:
- A slight body weight loss was noted in two males in the 500 mg/kg dose group and in two males and two females in the 2000 mg/kg dose group during the day 0 to 7 body weight period.
- Body weight gain was noted for all other animals during the test period.
Gross pathology:
- Thickened skin at the treated test site was observed externally at the 2000 mg/kg dose level (in addition to the in-life scores).
- Four incidences of cyst(s) on the oviduct(s) were observed; however, these findings were not considered to be significant since they are commonly found in rabbits of this strain.
- No significant treatment-related gross internal findings were observed at necropsy on study day 14.
Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study the percutaneous LD50 was found to be in excess of 2000 mg/kg for both male and female rabbits.
Executive summary:

The acute dermal toxicity of the test material was investigated in accordance with the standardised guidelines OECD 402, OPPTS 870.1200 and JMAFF 59 NohSan No. 4200, under GLP conditions.

The single-dose dermal toxicity of the test material was evaluated in New Zealand White rabbits. Initially, a limit test was performed in which one group of five male and five female rabbits received a single dermal administration of the test material at 500 mg/kg body weight. A second limit test was then performed in which one group of five male and five female rabbits received a single dermal administration of the test material at a dose of 2000 mg/kg body weight. Due to the severity of the dermal responses/clinical observations, for humane reasons no additional higher levels were attempted.

Following dosing, the limit test rabbits were observed daily and weighed weekly. A gross necropsy examination was performed an all animals at the time of scheduled euthanasia (day 14). Clinical abnormalities observed during the study included decreased defecation, soft stools, faeces small in size, decreased food consumption, breathing abnormalities and dark material around the facial area. Severe dermal irritation was noted at the site of test material application. Severe dermal irritation was noted for the 2000 mg/kg dose level including apparent haemorrhaging at the test site. A slight body weight loss was noted in two males in the 500 mg/kg dose group and in two males and two females in the 2000 mg/kg dose group during the day 0 to 7 body weight period. Body weight gain was noted for all other animals during the test period. No significant gross internal findings were observed at necropsy on study day 14.

Under the conditions of this study the percutaneous LD50 was found to be in excess of 2000 mg/kg for both male and female rabbits.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Additional information

Acute Oral Rat

The acute oral toxicity of the test material was determined in accordance with the standardised guidelines EPA OPPTS 870.1100 and JMAFF 59 NohSan No. 3850, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The single-dose oral toxicity of the test material was evaluated in Sprague-Dawley rats. An LD50 study was performed in which 5 groups of five male and five female rats received a single oral administration of the test material at graded dosage levels. Following dosing, the LD50 study rats were observed daily and weighed weekly. A gross necropsy examination was performed on all LD50 study animals at the time of death or scheduled euthanasia (day 14).

All male rats died at the 85 mg/kg dose. No males died at the 75 mg/kg dose. For females, all rats died at the 350 mg/kg high dose but none at the 50 mg/kg low dose. Partial deaths were observed at the intermediate doses. All mortality occurred by study day 2. The most notable clinical abnormalities observed during the study included breathing abnormalities, prostration, tremors, soft stools/few faeces, faecal stain, dilated pupils, skin pale in colour, ocular discharge, salivation, wobbly gait, decreased activity, eyelids partially closed, and dark material around the facial area. Body weight gain/maintenance was noted for all surviving animals during the test period. The most notable gross internal findings were observed in the animals that died and included abnormal content of the digestive tract, foci/thickening of the stomach, mottled and/or dark red lung lobes, foci on the thymus and blackish-purple lobes of the liver. No significant gross internal findings were observed at necropsy on study day 14.

Under the conditions of this study, the acute oral LD50 was determined to be 79.84 mg/kg and 131.98 mg/kg in male and female rats respectively. 

Acute Inhalation

The acute inhalation toxicity of the test material was investigated in accordance with the standardised guidelines EPA OPPTS 870.1300 and JMAFF 8147 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The acute inhalation toxicity was evaluated in a four hour, single-exposure study in rats. The test material was administered to four groups of five male and five female albino rats each via whole-body exposure as a vapour atmosphere at concentrations of 581, 710, 797 and 1189 ppm. Following each exposure, all surviving animals were maintained for a 14-day observation period. Parameters evaluated were mortality, clinical observations, body weights and gross necropsy.

Eight animals in each of the 710 and 797 ppm groups and all 10 animals in the 1189 ppm group died within two days of exposure as a result of test article administration. There were no deaths in the 581 ppm group.

Ataxia, clear nasal discharge, gasping, hypoactivity, laboured respiration, rales and red material around the nose were observed immediately following exposure in the 581 ppm group. Gasping, hypoactivity and/or laboured respiration were observed for the surviving animals in 710, 797 and 1189 ppm groups immediately following exposure. During the 14-day observation period, decreased defecation/urination, hypoactivity, red material on various body surfaces and rales were noted in the 581 ppm group. In addition to these clinical findings, gasping, mucoid faeces, laboured respiration and unkempt appearance were also observed for the surviving animals in the 710 ppm group. For the surviving animals in the 797 ppm group, hypoactivity and red material on various body surfaces were noted. All surviving animals were considered clinically normal by day 4 for the 710 and 797 ppm groups and by day 7 for the 581 ppm group.

Body weight losses were observed for all animals in the 581, 710 and 797 ppm groups from days 0 to 3. All surviving animals surpassed their initial day 0 body weight by day 14.

Macroscopic findings were limited to the animals that died early. In the 1189 ppm group, a distended, gas-filled stomach was noted for eight animals. For animals found dead in the 797 ppm group, a distended intestine (parts or entire length) was observed for three males and one female and dark red adrenal glands and a haemorrhagic thymus gland were each observed for two females. Dark red lungs were observed for two males and a reddened pituitary gland was observed for one male in the 710 ppm group. In addition, a distended, gas-filled stomach was observed for one male and a reddened glandular portion of the stomach was observed for two females in the 710 ppm group.

Under the conditions of this study, the acute inhalation LC50 was 691 ppm in both male and female rats.

Acute Oral Mouse

The acute oral toxicity of the test material was determined in accordance with the standardised guideline OPPTS870.1100, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The single-dose oral toxicity of the test material was evaluated in CD-1 mice. The study was initiated with a range-finding test at levels ranging from 50 to 1000 mg/kg body weight. Following the range-finding test, an LD50 study was performed in which groups of five male and five female mice received a single oral administration of the test material at graded dosage levels (five males and five females per level). Following dosing, the LD50 study mice were observed daily and weighed weekly. A gross necropsy examination was performed on all animals at the time of death or scheduled euthanasia (day 14).

All mortality occurred by study day 3. The most notable clinical abnormalities observed during the study included soft stools, slight to severe urine stain, slight to moderate faecal stain, decreased/no defecation, decreased food consumption, salivation, dilated pupil(s), eyelids partially closed, piloerection, rough haircoat, prostration, apparent hypothermia, decreased activity, breathing abnormalities, skin blue in colour - the entire body, hunched posture and wobbly gait. In the 100 mg/kg dose group, body weight loss was noted for one male and one female during the day 0-7 body weight interval and one male during the day 7-14 body weight interval. Two male and two female mice in the 175 mg/kg dose group lost body weight during the day 0-7 body weight interval. Two 200 mg/kg females lost body weight during the day 0-7 body weight interval and one female lost body weight during the day 7-14 body weight interval. In the 225 mg/kg dose group, two females lost

body weight during the day 0-7 body weight interval. Body weight gain or maintenance was noted for the remaining surviving animals during the test period. Significant gross internal findings observed in the animals found dead were abnormal content of the digestive tract, dilated pelvis of the kidneys, reddened glandular mucosa of the stomach and red fluid in the thoracic cavity. At scheduled euthanasia, the significant gross internal findings were thickened areas of the stomach associated with adhesion involving the body wall and stomach serosa.

Under the conditions of this test, the acute oral LD50 of the test material in the male mouse was determined to be 155 mg/kg. In the female mouse, the oral LD50 was determined to be 214 mg/kg. In the sexes combined, the oral LD50 was determined to be 179 mg/kg .

Acute Dermal

The acute dermal toxicity of the test material was investigated in accordance with the standardised guidelines OECD 402, OPPTS 870.1200 and JMAFF 59 NohSan No. 4200, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The single-dose dermal toxicity of the test material was evaluated in New Zealand White rabbits. Initially, a limit test was performed in which one group of five male and five female rabbits received a single dermal administration of the test material at 500 mg/kg body weight. A second limit test was then performed in which one group of five male and five female rabbits received a single dermal administration of the test material at a dose of 2000 mg/kg body weight. Due to the severity of the dermal responses/clinical observations, for humane reasons no additional higher levels were attempted.

Following dosing, the limit test rabbits were observed daily and weighed weekly. A gross necropsy examination was performed an all animals at the time of scheduled euthanasia (day 14). Clinical abnormalities observed during the study included decreased defecation, soft stools, faeces small in size, decreased food consumption, breathing abnormalities and dark material around the facial area. Severe dermal irritation was noted at the site of test material application. Severe dermal irritation was noted for the 2000 mg/kg dose level including apparent haemorrhaging at the test site. A slight body weight loss was noted in two males in the 500 mg/kg dose group and in two males and two females in the 2000 mg/kg dose group during the day 0 to 7 body weight period. Body weight gain was noted for all other animals during the test period. No significant gross internal findings were observed at necropsy on study day 14.

Under the conditions of this study the percutaneous LD50 was found to be in excess of 2000 mg/kg for both male and female rabbits.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as Category 3 in regards to acute oral toxicity, H301: Toxic if swallowed. It is also classified as Category 3 with regard to acute inhalation toxicity H331: Toxic if inhaled.

Although the available study investigating the acute dermal toxicity of the substance indicates that the substance should not be classified, the substance has a harmonized classification for this endpoint as Category 4. H312: Harmful in contact with skin and so this classification is also applied.