Ziram

Endpoint summary

Description of key information

Oral

Key, report number HRC 89690/UCB 315/AC, (rat): LD50 = 267 mg/kg bw (females) and 381 (males)

Inhalation

Key, report number UCB 314/89684 (rat): LC50 = 0.08 mg/L (males) and 0.06 mg/L (female)

Key, report number 378/2, (rat): LC50 = 0.18 mg/L (males) and 0.08 mg/L (females)

Dermal

Key, report number HRC 89338D/UCB 316/AC (rabbit): LD50 > 2000 mg/kg bw (males and females)

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:

until 10 Mar 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 142 - 158 g
- Fasting period before study: Rats were fasted the night before dosing.
- Housing: Five rats per cage of similar sex were housed in metal cages with wire mesh floors.
- Diet: Standard laboratory rodent diet (Biosure LAD 1), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 62
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 1% w/v

MAXIMUM DOSE VOLUME APPLIED: 20.0 mL/kg bw

DOSAGE PREPARATION:
Rats were dosed via gavage. Individual doses were calculated based on the animal’s body weight recorded just prior to dosing at a dose volume of 20 mL/kg bw. Rats were fasted the night before dosing and approximately 4 h after dosing.

CLASS METHOD
- Rationale for the selection of the starting dose: A trial test was carried out to establish a dosing regimen for the main study. Groups of 2 male and female rats were orally dosed at 200 and 800 mg/kg bw.

Doses:

250, 400, 640 mg/kg bw

No. of animals per sex per dose:

5/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for mortality, general condition and clinical signs (time, onset, severity and recovery) soon after dosing and at frequent intervals for the remainder of Day 1 (a period of 6 h) and two times a day on subsequent days for 14 days. Individual body weights were recorded prior to dosing (Day 1), on Day 8 and the day of necropsy.
(Day 15 or day of death).
- Necropsy of survivors performed: On Day 15, all surviving animals were euthanised by cervical dislocation. Gross pathological examinations, including external examination and opening of the abdominal and thoracic cavities, were conducted on all animals in the study.

Statistics:

The acute median lethal oral dose (LD50) to male and female rats was calculated using probit analysis.

Preliminary study:

The results of the preliminary study indicated that the acute medium lethal oral dose to male rats for the test substance was approximately 800 mg/kg bw and for females between 200 and 800 mg/kg bw. Therefore, in the main study oral doses of 250, 400 and 640 mg/kg bw were used.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

267 mg/kg bw

Based on:

test mat.

95% CL:

113 - 393

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

381 mg/kg bw

Based on:

test mat.

95% CL:

227 - 594

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

320 mg/kg bw

Based on:

test mat.

95% CL:

176 - 422

Mortality:

There were deaths amongst males and females dosed at all dose levels. Deaths occurred from Day 2 to Day 3.
250 mg/kg bw: 1 male and 2 females
400 mg/kg bw: 3 males and 4 females
640 mg/kg bw: 4 males and 5 females

Clinical signs:

other: pilo-erection, abnormal body carriage (hunched posture), abnormal gait (waddling), lethargy, decreased respiratory rate, ptosis, pallor of the extremities and diarrhoea

Body weight:

other body weight observations

Remarks:

Slightly low body weight gains were recorded for one male and one female dosed at 250 mg/kg bw and one male dosed at 640 mg/kg bw on Day 8. All other rats achieved anticipated body weight gains throughout the study.

Gross pathology:

Autopsy of rats revealed no macroscopic abnormalities.

Interpretation of results:

other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, classification for acute oral toxicity Category 3 (H301) is warranted.

Conclusions:

The GLP-compliant study was performed in accordance with EPA OPP 81-1. The test substance was administered by gavage to CD rats at dose levels of 250, 400 and 640 mg/kg bw. Under the conditions chosen, the acute oral LD50 value was determined to be 381 mg/kg bw for male and 267 mg/kg bw for female rats. According to criteria of the CLP Regulation (EU) No. 1272/2 008, classification of the test substance for acute oral toxicity category 3 (H301) is warranted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

267 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfill the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Version / remarks:

not specified

Deviations:

not specified

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, USA
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: approximately 200 g
- Housing: Animals were housed in groups of 5 per sex in polypropylene cages.
- Diet: Rodent standard diet (Biosure LAD 1), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 — 24 °C, except on 3 occasions when temperatures of 17 °to 17.5 °C were recorded
- Humidity (%): 28 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.3 - <= 2.7 µm

Geometric standard deviation (GSD):

>= 2.01 - <= 4.3

Remark on MMAD/GSD:

For detail on MMAD, please refer to Table 1 under "Any other information on results incl. tables".

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The whole-body exposure chambers used were of square section and were fitted with pyramidal tops. The chambers were made of perspex.
- Exposure chamber volume: 120 L
- Method of holding animals in test chamber: For exposure, the rats were held in cages of stainless steel mesh partitioned to provide 10 individual animal compartments.
- Source and rate of air (airflow): clean, dried, compressed air; flow rate of 25 L/min
- System of generating particulates/aerosols: A Wright dust generator was used to produce a test atmosphere containing the test substance. For all test groups the test atmosphere produced by the generator was passed through a glass elutriation column to reduce, by sedimentation, the amount of non-respirable particulate in the test atmosphere.
- Method of particle size determination: The particle size was determined by using an Andersen mini sampler. The collected material was weighed to determine the particle size distribution.
- Temperature, humidity: 22.0 - 24.3 °C, 41 - 57%

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: Five air samples, of 2.5 - 5 min duration, were taken from the chamber during each exposure. The samples were taken from a port in the wall of the chamber at the same level as, and near to, the breathing zone of the rats. Each air sample was withdrawn, at 4 L/min, through a weighed glass fibre filter mounted in an open-face filter holder. The volume of each air sample was measured with a wet-type gas meter.
- Time needed for equilibrium of exposure concentration before animal exposure : 11 min

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.020, 0.029, 0.047, 0.098 and 0.145 mg/L air

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All rats were observed continuously for signs of reaction to the test substance during exposure and at least twice daily throughout the observation period. All rats were weighed daily from the day of delivery until the end of the observation period.
- Necropsy of survivors performed: At the end of the 14-day observation period, the surviving rats were anaesthetised by intraperitoneal injection of pentobarbitone sodium and killed by exsanguination. All rats were subjected to a detailed macroscopic examination. The lungs were removed, dissected clear of surrounding tissue and weighed in order to calculate the lung weight to body weight ratio. The lungs were infused with, and preserved in, buffered 10% formalin together with samples of the liver and kidneys for possible macroscopic examination.
- Other examinations performed: The amount of food and water consumed by each cage of rats was measured daily from the day following arrival. The daily mean intakes of food and water for each rat were calculated from the recorded data.

Statistics:

Data were analysed statistically by the log probit method.

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

0.08 mg/L air

Based on:

test mat.

Remarks:

corresponding to 6.72 mg/kg bw

95% CL:

>= 0.052 - <= 0.828

Exp. duration:

4 h

Remarks on result:

other: corresponding to 6.72 mg/kg bw

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

0.06 mg/L air

Based on:

test mat.

Remarks:

corresponding to 5.04 mg/kg bw

95% CL:

>= 0.043 - <= 0.077

Exp. duration:

4 h

Remarks on result:

other: corresponding to 5.04 mg/kg bw

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.07 mg/L air

Based on:

test mat.

95% CL:

>= 0.059 - <= 0.081

Exp. duration:

4 h

Remarks on result:

other: corresponding to 5.88 mg/kg bw

Mortality:

0.020 mg/L: 0/5 males and 0/5 females
0.029 mg/L: 0/5 males and 0/5 females
0.047 mg/L: 0/5 males and 4/5 females
0.098 mg/L: 2/5 males and 4/5 females
0.145 mg/L: 5/5 males and 4/5 females

Clinical signs:

other: during exposure: partial closing of the eyes, irregular respiration rate, sallivation, pilo-erection and reduced activity; during observation period: abnormal respiration, a dark appearance of the eyes, lethargy and death (surviving animals recovered)

Body weight:

There were moderate to marked decreases of body weight or reductions in the rate of body weight gain for up to 2 days following exposure in all dose groups. Subsequently, weight gain for rats that survived exposure to the test material was similar to that of the control rats.

Gross pathology:

There were no macroscopic abnormalities in the control animals and in the rats that survived exposure to the test substance.
In the animals that died during the study, the ratio of lung to body weight was increased. In addition, the lungs of the majority of the rats that died were congested. A frothy fluid in the trachea, occasional swollen appearance of the lungs and gas-filled stomachs were found in a proportion of the desendentrats.

Table 1: Summary of chamber atmosphere conditions

Group	Meana chieved concentration of the test substance [mg/L air]		Particle size
	Mean	Variation [%]	MMAD [µm]	SD	≤5.5 µm [%]
Control	-	-	-	-	-
Treatment groups	0.020	45	2.7*	2.01	82.7
	0.029	66	1.8	3.43	83.7
	0.047	36	1.3	3.22	92.7
	0.098	26	1.4	3.02	90.6
	0.145	79	2.3	4.30	76.4

MMAD:Mass median aerodynamic diameter
SD: Geometric standard deviation
* In view of the small amount of material collected in the sample for this group, the calculated MMAD and SD should be interpreted with caution. The fact that there was no measureable amount collected on the filter will have caused the calculated MMAD to be larger than the true value.

Food consumption

Food consumption was reduced for up to 2 days following exposure to the test material.

 

Water consumption
Water consumption was reduced for up to 2 days following exposure. There were some instances of high water consumption during the observation period for female rats exposed to the test substance.

Interpretation of results:

other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, classification for acute oral toxicity Category 2 (H330) is warranted.

Conclusions:

The study was performed according to EPA OPP 81-3 (acute inhalation toxicity) and GLP. In this test, five male and female rats were exposed (whole-body) to the test substance at exposure levels of 0.020, 0.029, 0.047, 0.098 and 0.145 mg/L of air for 4 h. Under the conditions of the test, the acute inhalation LC50 value was determined to be 0.08 mg/L air for male and 0.06 mg/L air for female rats. According to criteria of the CLP Regulation (EU) No. 1272/2008, classification of the test item for acute inhalation toxicity category 2 (H330) is warranted.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Oct - 05 Nov 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP guideline study with minor deviations (Animals received partially different exposure concentrations depending on sex)

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted in 1981

Deviations:

yes

Remarks:

Animals received partially different exposure concentrations depending on sex.

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

not specified

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Version / remarks:

not specified

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kent, UK
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 244 - 310 g (males), 221 - 276 g (females)
- Housing: Animals were housed in groups of 5 by sex in suspended polypropylen cages with stainless steel lids furnished with softwood sawdust.
- Diet: Rodent standard diet (Rat and Mouse Expanded Diet No. 1, Special Diet Services Limited, Essex, UK), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: a t least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 22
- Humidity (%): 40 - 68
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.2 - <= 1.7 µm

Geometric standard deviation (GSD):

>= 0.48 - <= 0.56

Remark on MMAD/GSD:

The mean mass median aerodynamic diameter (MMAD) ranged between 1.2 and 1.7 µm and the geometric standard deviation (GSD) ranged between 0.48 and 0.56. The Particle size analysis indicated that a significant portion of the inhaled atmosphere reached the lungs (> 88% with diameter < 4 µm). For details, please refer to the attached background material 1.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Cylindrical exposure chamber
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: each rat was held in a tapered, polycarbonate restrained tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber O-ring.
- Source and rate of air (airflow): compressed air was supplied by means of an oil free compressor and was passed through a water trap and respiratory quality filter, 20 L/min
- System of generating particulates/aerosols: A dust atmosphere was produced from the test material using a wright dust feed mechanism located at the top of the exposure chamber and driven by a variable speed motor.
- Method of particle size determination: Particle size was determined at least twice during the exposure period by a cascade impactor.
- Temperature, humidity, pressure in air chamber: 17 - 23 °C, 42 - 70% relative humidity, oxygen content was at least 19%

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: The chamber concentration was estimated at regular intervals (at least every 15 min) during exposure period by gravimetric method.
- Samples taken from breathing zone: yes
- Time needed for equilibrium of exposure concentration before animal exposure : 7 min

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.06 (females only), 0.10 (males and females), 0.52 (males only) and 0.96 mg/L air (males and females)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All rats were observed for signs of reaction to the test substance at hourly intervals during the exposure, 1 h after termination of the exposure and subsequently once daily for 14 days. Individual body weights were recorded on the day of exposure and on Days 7 and 14 or at death.
- Necropsy of survivors performed: At the end of the 14-day observation period, surviving rats were euthanised by intravenous overdose of sodium pentobarbitone. All rats were subjected to a full external and internal examination and macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Statistics:

Data were analysed by the method of Thompson (1947) to calculate the acute inhalation median lethal concentration (LC50).

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

0.18 mg/L air

Based on:

test mat.

95% CL:

>= 0.1 - <= 0.31

Exp. duration:

4 h

Remarks on result:

other: corresponding to 15.13 mg/kg bw

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

0.08 mg/L air

Based on:

test mat.

95% CL:

>= 0.07 - <= 0.1

Exp. duration:

4 h

Remarks on result:

other: corresponding to 6.72 mg/kg bw

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

0.13 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: corresponding to 10.93 mg/kg bw

Mortality:

0.06 mg/L air: 0/5 females
0.10 mg/L air: 0/5 males and 4/5 females (one female was sacrificed in extremis 2 h after completion of the exposure, three further females died on Day following exposure)
0.52 mg/L air: 5/5 males (3hree males died 120 to 150 min after start of exposure and one died 50 min after completion of exposure, the remaining male was sacrificed in extremis on Day 1 following exposure)
0.96 mg/L air: 5/5 males and 5/5 females (all animals died or were sacrificed in extremis within 120 to 183 min after the start of exposure)

Clinical signs:

other: please refer to "Any other information on results incl. tables"

Body weight:

Surviving animals exposed to 0.10 mg/L air and the females exposed to 0.06 mg/L air showed expected body weight gain throughout the study.

Gross pathology:

All animals exposed to 0.96 mg/L air showed lungs that were haemorrhaged and swollen. One female killed in extremis also showed abnormal redness of the lungs. In several animals reddening of the small intestine was noted. The liver of one female killed in extremis appeared dark.
In the males exposed to 0.52 mg/L air haemorrhage and swollen appearance of the lungs were again common. One male killed in extremis showed lungs that were abnormally red, swollen and fluid filled with dark patches. Isolated incidents of dark liver and gaseous distension of the small intestine were also noted.
Following exposure to 0.10 mg/L air one female killed in extremis showed clear fluid present around the snout and mouth. The lungs of this animal also appeared haemorrhagic, swollen and fluid filled. Three females that died in this group showed abnormal redness and swollen appearance of the lungs and congestion of the small intestine. The livers of two females appeared dark. No abnormalities were detected in surviving animals.
No abnormalities were observed in the females exposed to 0.06 mg/L air at necropsy.

Clinical signs

During exposure to 0.96 mg/L air animals showed wet fur and decreased respiratory rate. Hunched posture, lethargy, pilo-erection, gasping respiration, ataxia and ptosis were also noted in three animals that were removed from the chamber 183 min after the start of the exposure.

The males exposed to 0.52 mg/L air showed decreased respiratory rate during exposure. Signs of wet fur, hunched posture, lethargy, pilo-erection, ataxia, ptosis, laboured respiration and pallor of the extremities were noted in two males on removal from the chamber. Additional signs of gasping respiration, noisy respiration and increased salivation were also noted in the one surviving male one hour after completion. On day one additional signs were evident including hypothermia, pallor of the extremities and red/ brown stains around the snout.

During exposure to 0.10 mg/L air wet fur and decreased respiratory rate were apparent in many animals. On removal from the chamber all animals showed wet fur and decreased respiratory rate in addition to hunched posture and pilo-erection. Signs of ataxia were noted and one male showed ptosis. Similar signs were noted one hour after completion of exposure with the exception of wet fur only being evident in one female. Isolated incidents of lethargy, laboured respiration, pallor of the extremities and red/brown stains around the snout were noted and animals showed ptosis. On day one following exposure all surviving animals showed hunched posture, pilo-erection and decreased respiratory rate, one male showed red/brown stains around the snout and lethargy was noted in the female survivor. On day two three males appeared normal but noisy respiration was noted in the female. All surviving animals appeared normal by day 7.

The females exposed to 0.06 mg/L air showed wet fur and decreased respiratory rate during exposure. On removal from the chamber hunched posture, lethargy, pilo-erection and ptosis were noted and two females showed ataxia. On day one following exposure there were still signs of hunched posture, pilo-erection and decreased respiratory rate and an incident of red/brown stains around the snout. On day two all females appeared normal.

Interpretation of results:

other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, classification for acute inhalation toxicity Category 2 (H330) is warranted.

Conclusions:

The study was performed according to OECD guideline 403 and GLP. Five male and female rats were exposed (nose only) to the test substance at exposure levels of 0.06 (females only), 0.10, 0.52 (males only) and 0.96 mg/L of air for 4 h. Under the conditions of the test, the acute inhalation LC50 value was determined to be 0.18 mg/L air for male and 0.08 mg/L air for female rats. According to criteria of the CLP Regulation (EU) No. 1272/2008, classification of the test item for acute inhalation toxicity category 2 (H330) is warranted.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

0.06 mg/L air

Physical form:

inhalation: dust

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus, sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of 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:

08 Feb - 22 Feb 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-2 (Acute Dermal Toxicity)

Version / remarks:

not specified

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

not specified

Deviations:

yes

Remarks:

24 h exposure

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
- Source: A. Smith, Warlingham, Surrey and Froxfield Rabbits, Hampshire, UK
- Age at study initiation: 9 - 13 weeks
- Weight at study initiation: 2.2 - 2.9 kg
- Housing: individually in metal cages with perforated floors
- Diet: Standard laboratory diet (Stanrab P), ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 22
- Humidity (%): 49
- Air changes (per hr): approximately 19
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal-lumbar area
- % coverage: equivalent to 10% of the total body surface
- Type of wrap if used: The prepared area was covered with gauze which was held in place with an impermeable dressing encircled firmly around the trunk.

REMOVAL OF TEST SUBSTANCE
- Washing: The residual test substance was removed at the end of the 24-h exposure period by washing with water and blotting dry with absorbent paper.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 g/kg bw
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
No vehicle was used.

Duration of exposure:

24 h

Doses:

2 g/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for mortality and clinical signs soon after dosing and then at frequent intervals for the reminder of Day 1. On subsequent days, the animals were observed at least twice for 14 days. Individual body weights were recorded prior to dosing (Day 1), on Day 8 and the day of necropsy (Day 15).
- Necropsy of survivors performed: On Day 15, all surviving animals were euthanised by an intravenous overdose of pentobarbitone sodium. Gross pathological examinations, including external examination and opening of the abdominal and thoracic cavities, were conducted on all animals in the study. The macroscopic appearance of abnormal organs was recorded.

Statistics:

No statistical analysis was performed.

Preliminary study:

A trial test was carried out by dosing one male and one femlae rabbit with 2 g/kg bw. The results of the preliminary study indicated that the acute median lethal dermal dose to male and female rabbits of the test substance was greater than 2 g/kg bw.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

No deaths occured.

Clinical signs:

other: No signs of systemic toxicity were observed in response to treatment.

Body weight:

lower than 10% body weight loss

Remarks:

A loss in body weight was recorded for two females on Day 8. All other rabbits achieved anticipated body weight gains throughout the study.

Gross pathology:

Pale renal cortices were observed in the kidneys of one male at post-mortem. Terminal autopsy revealed no other macroscopic abnormalities.

Other findings:

Dermal responses:
Slight erythema only was observed in two males and two females. The reactions had resolved completely by Day 3 or 4 of the study. The remaining three males and three females showed no response to treatment.

Interpretation of results:

other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, no classification is required.

Conclusions:

The study was performed according to EPA OPP 81-2 (Acute Dermal Toxicity) under GLP conditions. Male and female rabbits of the New Zealand White strain received the test substance at a single dose of 2000 mg/kg bw, moistened with destilled water. Under the conditions chosen, the acute dermal LD50 was determined to be > 2000 mg/kg bw for male and female rabbits. According to criteria of the CLP Regulation (EU) No. 1272/2008, no classification of the test item for acute dermal toxicity is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus, sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

To assess acute toxicity of the test substance, data after oral, inhalation and dermal exposure to the test substance are considered.

Acute toxicity: oral

In a GLP-compliant study (Report number: HRC 89690/UCB 315/AC), which was performed in accordance with EPA OPP 81-1 groups of 5 male and female rats were orally dosed with 250, 400 and 640 mg/kg bw of the test substance. Under the conditions chosen, the acute oral LD50 value was determined to be 381 mg/kg bw for male and 267 mg/kg bw for female rats. According to criteria of the CLP Regulation (EU) No. 1272/2 008, classification of the test substance for acute oral toxicity category 3 (H301) is warranted. This is in line with the harmonized classification (minimum classification as acute toxic 4*, H302).

Acute toxicity: inhalation

A GLP-conform acute inhalation toxicity study was performed according to EPA OPP 81-3 and under GLP and was therefore considered as key study (Report number: UCB 314/89684). The purpose of this study was to assess the acute inhalation toxicity of the test substance following a 4-h exposure of male and female rats. In this test, five male and female rats were exposed (whole-body) to exposure levels of 0.020, 0.029, 0.047, 0.098 and 0.145 mg/L air, respectively, for 4 h. Animals were observed continuously for clinical signs during the 4 h exposure period. After that, rats were then observed twice a day for morbidity and mortality for a period of 14 days. Individual body weights were recorded daily. All animals were subjected to gross necropsy. All rats survived in the exposure groups of 0.020 and 0.029 mg/L air. In the group exposed to 0.047 mg/L, all males survived but 4 out of 5 females died. In the group exposed to 0.098 mg/L, 2 males and 4 females died. In the group exposed to 0.145 mg/L, all 5 males died as well as 4 out of 5 females. Reduced body weight or rate of body weight gain for up to 2 days following exposure to the test substance was observed. Similarly, the food and water consumption was reduced for up to 2 days following exposure. There were some instances of high water consumption during the observation period for female rats exposed to the test substance. The mean mass median aerodynamic diameter (MMAD) ranged from 1.3 to 2.7 µm and the geometric standard deviation (GSD) ranged from 2.01 to 4.30 µm. The median lethal concentration (LC50 value) for the test substance in the rat was 0.08 (6.72 mg/kg bw) mg/L in male rats and 0.06 (5.04 mg/kg bw) mg/L in female rats. The combined LCs was 0.07 mg/L (5.88 mg/kg bw).

These results were supported by a further GLP-conform acute inhalation toxicity study (key study), which was performed according to EPA OPP 81-3 and OECD 403 (Report number: 378/2). In this key study, 5 male and female rats were exposed (nose only) to the test substance at dose levels of 0.06 (females only), 0.10, 0.52 (males only) and 0.96 mg /L air, respectively, for 4 h. Under the conditions of the test, the acute inhalation LC50 value was determined to be 0.18 mg/L air for male and 0.08 mg/L air for female rats.

A third acute inhalation toxicity study is only mentioned here for the purpose of data completeness. In this study (Report number: 12101), rats were exposed to the test substance (nose-only exposure) at dose levels of 0.2, 1.15 and 4.96 mg/L air for 4 h. The acute inhalation LC50 value was determined to be lower than 0.2 mg/L.

In conclusion, based on the available data on inhalation toxicity of the test substance and according to criteria of the CLP Regulation (EU) No. 1272/2008, classification of the test item for acute inhalation toxicity category 2 (H330) is warranted, which is in line with the harmonized classification. Further, respiratory irritation was observed after single inhalation exposure of rats (Report number: UCB 314/89684 and 378/2) (for details, please refer to the respective study records and the endpoint discussion provided under “Irritation”). In conclusion, classification for STOT-SE 3 (H335) is warranted according to the classification criteria of the CLP Regulation (EU) No. 1272/2008.

Acute toxicity: dermal

A GLP-conform acute dermal toxicity study was performed according to EPA OPP 81-2 (Report number: HRC 89338D/UCB 316/AC), which is considered as key study. A limit dose of 2000 mg/kg bw was topically applied for 24 h to dorsal-lumbar area of 5 male and female rabbits under occlusive dressing. Under the conditions chosen, the acute dermal LD50 was determined to be > 2000 mg/kg bw for male and female rabbits. According to criteria of the CLP Regulation (EU) No. 1272/2008, no classification of the test item for acute dermal toxicity is required.

Justification for classification or non-classification

The available data on acute oral toxicity and acute inhalation toxicity meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore sufficient for classification of the test substance for acute oral toxicity category 3 (Acute Tox. 3, H301) and acute inhalation toxicity category 2 (Acute Tox. 2, H330). Further, the test substance is a respiratory irritant as indicated by the results of the acute and 28-d inhalation toxicity study and is classified accordingly (STOT SE3, H335 (CLP)). Based on the available data on acute dermal toxicity, no classification of the test substance for acute dermal toxicity is required.

The substance is listed in Annex VI of Regulation (EC) 1272/2008 (006-012-00-2). Harmonised classification in regard to minimum classification was adopted according to the available experimental data if appropriate.