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Key value for chemical safety assessment

Acute toxicity: via oral route

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Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 21 - August 19, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD test guidelines and GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino breeding center)
- Age at study initiation: 9 weeks
- Weight at study initiation: 186.1-198.8 g
- Fasting period before study: From the evening on the day before the administration (approx 18 hours predose) to about 3 hours postdose.
- Housing: Stainless steel cages (W226xD346xH198 mm), stainless steel racks and feeders, one animal per cage
- Diet (ad libitum): Pellet diet (CRF-1, Oriental Yeast Co., Ltd.) (each lot analysed: contaminants confirmed to be within acceptable limits established by the test facility)
- Water (ad libitum): Well water (analysed every 6 months: contaminants confirmed to be within acceptable limits in compliance with waterworks law, Japan)
- Acclimation period: 6 days for experiment 1 and 8 days for experiment 2

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.6-24.6
- Humidity (%): 44.9-61.3
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: test substance solved to make a dosing solution at a concentration of 100 mg/ml

MAXIMUM DOSE VOLUME APPLIED: 20 ml/kg

DOSAGE PREPARATION: The dosing volumes for individual animals were calculated on the basis of the body weight measured just before administration.

CLASS METHOD
- Rationale for the selection of the starting dose: The lethal dose of a similar substance was reported to be 2000 mg/kg or above. Therefore, the dose level for the first administration was set at 2000 mg/kg, which is the maximum dose recommended in the guideline applied to this study. As a result, as no death or moribundity was observed on the day after the first administration the dose level for the second administration was also set at 2000 mg/kg.
Doses:
2000 mg/kg
No. of animals per sex per dose:
3 females in each of 2 experiments
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality and clinical signs were observed before the dosing (Day 1, day of initial administration), 30 min, 1, 3, and 5 hours postdose on Day 1 and once daily for 14 days thereafter. Body weight was measured just before the administration (Day 1) and on Days 4, 8, and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: none
Statistics:
not applicable
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No deaths were noted in any animal in experiment 1 and 2.
Clinical signs:
other: Test substance mixed feces was observed in all animals from 3 hours on Day 1 to Day 3 or 4, and chromaturia (bluish) was observed in all animals on Day 2 and 3 in experiment 1 and 2. These changes were attributed to the test substance as this is a blue po
Gross pathology:
No abnormalities were noted in any animal in experiment 1 or 2.
Interpretation of results:
GHS criteria not met
Conclusions:
The LD50 of E-C104 in rats was estimated to be 2000 mg/kg or above under the conditions of this study (OECD 423).
Executive summary:

A single oral administration of E-C104 (dissolved in water) by gavage was conducted to female 9 weeks old Crl:CD(SD) rats to evaluate its acute oral toxicity in accordance with the OECD test guideline 423. A dose of 2000 mg/kg was employed for the first and second administration to each three rats. As a result, there was no mortality, body weight gain effects, or necropsy findings. Clinical signs were restricted to test substance mixed feces and chromaturia (bluish). In conclusion, the LD50 of E-C104 in rats was estimated to be 2000 mg/kg or above under the conditions of this study.

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

Acute toxicity: via inhalation route

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Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
aug-oct, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MAFF in Japan (12-Nousan-No. 8147, 2-1-3, 2000)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba Breeding Center (Ibaraki, Japan) of Charles River Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 310-331 grams (males); 199-216 grams (females)
- Fasting period before study: not indicated
- Housing: wire-mesh stainless steel cage; during quarantine and acclimatization period, 5 or 2 animals of the same sex were housed in a cage. After selection of animals, 5 animals of the same sex were housed in a cage.
- Diet: pellet diet MF (Oriental Yeast Co., Ltd., Tokyo); ad libitum
- Water: local tap water; ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 60±20
- Air changes (per hr): 8
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 23, 2011 To: September 27, 2011
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Animals were individually held in animal holders attached to a snout-only exposure chamber (total volume 31.2 L). The dust was generated by a turn-table type dust feeder with compressed air. The compressed air was supplied to the dust feeder through an air filter. Airflow to the chamber was controlled by an ejector in the dust feeder at a rate of 20 L/min. The chamber air was exhausted through an air filter system consisting of a bag filter, a HEPA filter and an activated charcoal filter. The differential pressure in the chamber was adjusted to and maintained between -20 and -5 mm H2O using exhaust-adjusting valve. Aiflow rate in the dust feeder was continuously confirmed and recorded every 30 minutes during the exposure period.

Nominal concentration: the total amount of active ingredient of the test substance supplied to the chamber was determined gravimetrically with adjustment for the purity. The nominal concentration (mg/L) was calculated by dividing the total amount of active ingredient (mg) by total air volume (L) deliverd during exposure.
Actual concentration: the actual concentration was quantified at 1, 2 and 3 hours after the initiation of exposure. Six litters of chamber air was drawn from the chamber sampling port. The test substance dust was trapped on a pre-weighed glass fiber filter and the amount of the test substance trapped was determined gravimetrically using an electronic balance. The actual concentration (mg/L) was calculated by dividing the amount of the active ingredient (mg) with adjustment for the purity trapped on a glass fiber filter by air volume (L) sampled.

TEST ATMOSPHERE (if not tabulated)
high dose
- Particle size distribution (MMAD): 4.49 µm (4.42-4.60 µm)
- GSD (Geometric st. dev.): 2.34 (2.24-2.51)
- Mean value of actual concentration = >4.99 mg/L (>4.68->5.18)
low dose
- Particle size distribution (MMAD): 4.28 µm (4.12-4.38 µm)
- GSD (Geometric st. dev.): 2.11 (2.09-2.12)
- Mean value of actual concentration = >2.02 mg/L (>1.94->2.11)
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5 mg/L (m, f), 2 mg/L (m)
No. of animals per sex per dose:
high dose: 5m, 5f
low dose: 5m
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observation for mortality and clinical signs at 2 hours after initiation of exposure, immediately, 1 and 4 hours after the exposure, and once daily thereafter until termination of the observation period. Each animal was weighed shortely before the exposure on the day of treatment and when found dead, on 1, 3, 7 and 14 days after exposure.
- Necropsy of survivors performed: yes
- Other examinations performed: none
Statistics:
The range of LC50 value was determined from the mortality.
Preliminary study:
A dose-finding acute inhalation toxicity study with 1.0 and 5.0 mg/L (E-C104) was performed with 5 rats/sex (study no IET 11-0059). In the 1 mg/L group no mortality was observed during the 7-day observation period, whereas in the 5 mg/L group 1 male and 1 female died at 1 day after exposure. Based on these results, a limit test at 5 mg/L was considered for the main study.
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
In the 5 mg/L group 1 male died 4 hours after exposure. No mortality was observed in the 2 mg/L group.
Clinical signs:
other: No clinical signs were observed in the low dose group. In the high dose group, sedation, deep respiration and labored respiration until one day after exposure, and disappeared on day 2 after exposure.
Body weight:
In the low dose group, body weight lossess were noted in all animals on day 1 after exposure, in 3 males on day 3 after exposure and in 1 male on day 7 after exposure (as compared with body weight before exposure).
In the high dose group, body weight losses were noted in all animals on day 1 after exposure and in 4 males on day 3 after exposure (as compared with body weight before exposure).
All animals gained their body weights at the end of the observation period (14 days after exposure).
Gross pathology:
No macroscopic abnormalities were noted in any surviving animal at necropsy at the end of the 14-day observation period.
Necropsy of the dead animal in the high dose group showed red color of the lung and foamy fluid in the trachea.
Other findings:
no
Interpretation of results:
GHS criteria not met
Conclusions:
In the 5 mg/L group, 1 male died on day 1 after snout-only exposure to E-C104 during 4 hours. Based on these results, the LC50 of E-C104 for rats in this study was more than 5 mg/L, and E-C104 needs not to be classified for acute inhalation toxicity according to GHS.
Executive summary:

The acute inhalation toxicity of E-C104 was tested in Sprague-Dawley rats. Animals were exposed for 4 hours in a snout-only exposure chamber to 5 mg/L (5m, 5f) or 2 mg/L (5m). MMAD and GSD were 4.49 µm and 2.34, respectively (high dose), and 4.28 µm and 2.11, respectively (low dose). In the low dose group, no mortality was observed, whereas 1 male rat died in the high dose group the day after exposure. No clinical signs were observed in the low dose group. In the high dose group, sedation, deep respiration and labored respiration until one day after exposure, which disappeared on day 2 after exposure. In the low dose group, body weight losses were noted in all animals on day 1 after exposure, in 3 males on day 3 after exposure and in 1 male on day 7 after exposure (as compared with body weight before exposure). In the high dose group, body weight losses were noted in all animals on day 1 after exposure and in 4 males on day 3 after exposure (as compared with body weight before exposure). All animals gained their body weights at the end of the observation period (14 days after exposure). No macroscopic abnormalities were noted in any surviving animal at the end of the observation period. Based on these results, The LC50 of E-C104 for both sexes of Sprague-Dawley rats in this study is therefor more than 5 mg/L.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LC50
Value:
5 mg/m³ air

Acute toxicity: via dermal route

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Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 20 - February 15, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to current OECD test guidelines and GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino breeding center)
- Age at study initiation: 7 week old males and 10 week old females
- Weight at study initiation: 201.5 to 219.4 g for males and 218.2 to 240.3 g for females
- Fasting period before study: not applicable
- Housing: Stainless steel cages (WxDxH: 226x346x198mm), stainless steel cage racks and feeders, one animal per cage.
- Diet (ad libitum): Autoclave-sterilised pellet diet (CRF-1, Oriental Yeast Co., Ltd.). Each lot has been analysed: contaminants confirmed to be within acceptable limits established by the test facility.
- Water (ad libitum): Well water admixed with NaClO (about 2 ppm). Analysed twice a year: contaminants confirmed to be within acceptable limits establised by the test facility.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.9-23.8
- Humidity (%): 48.4-57.0
- Air changes (per hr): 10 to 20
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Remarks:
moistened with water (1 ml)
Details on dermal exposure:
TEST SITE
- Area of exposure: 4 x 5 cm
- % coverage: within 10% of total body surface
- Type of wrap if used: After hair clipping animals were fitted with a neck collar. The test substance was applied on a lint cloth lined with an impermeable sheet (Blenderm, 3M Health Care). This was covered with an elastic adhesive bandage (SILKYTEX white No.5, Alcare Co. Ltd.)

REMOVAL OF TEST SUBSTANCE
- Washing: wiped with a gauze sheet immersed in lukewarm water and water was removed with dry tissue paper. Animals were fitted with neck collars during dosing and the recovery period.
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Required amount, calculated based on body weight on day of dosing. The lint cloth, where the substance was applied to, was moistened with an appropriate amount of water (1 ml).
Duration of exposure:
24 hours
Doses:
2000 mg/kg
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for their mortality and clinical signs before the dosing and at 30 min, 1, 3 and 6 hours postdose on the day of dosing (Day 1) and once daily for 14 days thereafter. All animals were subjected to body weight measurement before the dosing (Day 1) and on Days 4, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: no
Sex:
male/female
Dose descriptor:
LD50
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No death occured in either sex.
Clinical signs:
other: No substance related changes were seen in clinical signs.
Gross pathology:
No abnormalities were seen in either sexe.
Other findings:
No skin irritaion was observed.
Interpretation of results:
GHS criteria not met
Conclusions:
The dermal LD50 of E-C104 in rats was considered to be more than 2000 mg/kg under the conditions of this study (OECD 402).
Executive summary:

E-C104 was administered once percutaneously to five male and five female Crl:CD(SD) rats at a dose of 2000 mg/kg to evaluate its acute dermal toxicity in accordance with OECD test guideline 402. The dose was appllied for 24 hours occlusively after which the application site was wiped. As a result, no mortality occurred in either sex and no test substance related change was seen in clinical signs, body weight or necropsy. Therefore, the LD50 value of E-C104 in rats was considered to be more than 2000 mg/kg under the conditions of this study.

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

Additional information

Justification for classification or non-classification