Registration Dossier

Administrative data

Description of key information

Acute Oral Toxicity:

The test substance, ESA, was administered in single dose to female Sprague-Dawley (SD) to evaluate the lethal dose 50 (LD50) and acute toxicity. A starting dose level was set as 300 mg/kg because the toxicological information of the test substance was not known. During study period, mortalities, clinical signs and body weight changes and gross findings at necropsy were examined.

In the first and second steps (300 mg/kg), No mortalities and unusual clinical signs were observed during the observation period. In body weights, there were no changes related with the test substance. And abnormal gross findings were not detected in necropsy at the termination of observations.

In the third step (2,000 mg/kg), all animals died on the day of dosing (day 0) and day 1. Crawling position was observed in all animals at 30 minutes after administration.

No. 8 animal died immediately. No. 9 animal showed coma and terminal respiration at 1 hour and died at 2 hours after administration. No. 7 animal had crouching position and piloerection at 1 hour after administration and piloerection persisted on day 0. The animal died on day 1 finally. Necropsy of the decedents revealed the following; In animals died on day 0, glandular stomach was coated with black material and area of liver where is adjacent to stomach was discolored with grey-green. In an animal died on day 1, gastric filling with black substance and black coating in glandular stomach were founded in stomach and area of liver where is adjacent to stomach was discolored with light brown.

Therefore, the test substance ESA is considered to be classified as GHS category 4 (300 < LD50 cut-off ≤ 2,000 mg/kg b.w.) under present study conditions.

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:
Study initiation: 10 January 2011; Submission of final report: 14 March 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
Test type:
acute toxic class method
Limit test:
no
Specific details on test material used for the study:
Product name: ESA
Chemical name: 1,3,2-Dioxathiolane, 2,2-dioxide
Received date: 19 November 2010
Appearance: Straw colored solid
Purity: 99.1 %
Storage condition: refrigeration (recommended temperature: 2-8ºC)

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Species and strains: Specific Pathogen Free (SPF) Sprague-Dawley (SD) rats
Supplier: DooYeol Biotech (91, Baumoe-ro, Seocho-gu, Seoul, 06751, Korea)
Producer: Koatech (Jinwi-ro, Jinwi-myeon, Pyeongtaek-si, Gyeonggi-do, 17711, Korea)
Reason for selection of the species:
SD rats have been applied widely in general toxicity tests as a suitable experimental animal for toxicity testing. In addition, sufficient raw data has been accumulated
and is available for interpretation and evaluation of study results.
Date of acquisition:
1st: 11 January 2011
2nd: 24 January 2011
Sex and number of animals received:
1st: 7 female
2nd: 7 female
Age of animals received : 7 weeks old
Body weights on arrival :
1st: 167.67 ~ 178.95 g
2nd: 158.31 ~ 162.90 g
Quarantine and acclimation:
Animals were acclimated for 7-15 days after arrival. Only animals with the best appearance were selected for the test after observation during the acclimation period.
Animals were accepted based on the certification provided by the supplier

Age at the initiation of the administration : 8-9 weeks old
Body weights at the administration/;
1st 300 mg/kg dose group: 170.76 ~ 175.93 g
2nd 300 mg/kg dose group: 183.61 ~ 184.67 g
3rd 2,000 mg/kg dose group: 170.48 ~ 172.91 g
Number of animals administered:
Three female rats were used in each step. The test substance was administered to a total of nine female rats.

Grouping:
Animals were weighed one day before the test substance administration and grouped ramdomly according to body weight.
Identification of animals:
Individual animals were identified by tail marking with an oily-ink felt-pen and individual cages were distinguished by the individual card labeling. The information
of the test including study number, client, duration of the SPF room use, name of the study director, and names of technical assistance was listed in the record sheet.
It was provided at the entrance of the SPF animal room.
Disposal of remaining animals: They were euthanized.

Environmental and Housing Condition
Animal care room : Room 2 in the SPF animal facility area
Temperature and humidity : 22.0-22.6ºC and 40.9-44.3%RH
Ventilation frequency : 10-15 air changes/hour
Lighting cycle : 12 hours duration (lighting on at 8 a.m. and off at 8 p.m.)
Lighting intensity : 274 Lux
Noise : 54.8 dB
Concentration of ammonia : Less than 5 ppm
Type (size) of cages:
All animals were housed in stainless steel wire mesh cages (250W × 350L × 180H mm) during quarantine, acclimation, administration and observation period.
Number of animals per cage:
Not more than 3 animals during acclimation, administration and observation period.
Replacement for cages: at grouping
Feeds:
Type: solid laboratory animal feed
Supplier: DooYeol Biotech
Producer: Harlan
Feeding method : ad libitum using feeder
Contamination examination
The feed certification which was provided from the supplier was referred to examine contamination.
Water:
Type: Tap water purified by reverse osmosis filtering system
Feeding method: ad libitum using polycarbonate water bottles
Contamination analysis
Water certification from nationally certified inspection organization was referred to examine contamination
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Justification for the vehicle choice
According to the solubility test, the test substance was not dissolved in sterilized distilled water, corn oil and 0.5 % CMC (carboxy methyl cellulose). However, the test substance was dissolved in sterilized distilled water when it was heated in boiling water at 40 ºC.

Preparation of the test solution
The test substance was weighed accurately and dissolved in sterilized distilled water to prepare the test solution. A stability and homogeneity stability of the test solution was not performed because they were prepared and administerd freshly just prior to the administration. For the first and second steps (300 mg/kg), 0.31 g of the test substance was dissolved in sterilized distilled water to make a total volume of 10 ml.
For the third step (2,000 mg/kg), 2.02 g of the test substance was dissolved in sterilized distilled water to make a total volume of 10 ml.

Doses:
300 and 2000 mg/kg
No. of animals per sex per dose:
3 females per dose
Control animals:
no
Details on study design:
Methods of the administration
All rats were fasted one night before the administration day to empty their stomach. An intubation cannula was used for the oral administration. All fasted test animals were fed approximately at three to four hours after the test substance administration.

Calculation of dosing volume
Individual dosage was adjusted based on the fasted body weight measured right before the administration. Dosing volume was 10 ml/kg body weight.
Frequency and duration of the administration: Single dose

Determination of dose levels
Dose levels were determined in accordance with the OECD Guidelines for the Testing of Chemicals No. 423 ‘Acute Toxic Class Method’ (Adopted 17th Dec., 2001). and National Institute of Environment Research [Notice No. 2010-29]. A starting dose level was set as 300 mg/kg because the toxicological information of the test substance was not known.

Study procedure
At starting dose, 300 mg/kg, there were no mortalities. A dose level of second step was also set as 300 mg/kg.
The third step was performed with 2,000 mg/kg dose level because there were no moribund or mortalities in the second step. In the third step, all animals died by the test substance and the fourth step was not conducted. The dose level of next steps were designed after observing mortalities in each steps at least for 6 days.

Observations and Examinations
- Clinical signs and mortalities
General clinical signs or mortalities of all treated animals were observed once a day. On the day of dosing (day 0), all animals were observed right after administration, and then observed regularly at one hour intervals until six hours.
Animals were observed up to day 14 after the administration.
- Body weight measurement
Individual animals were weighed at the date of acquisition, at grouping, prior to administration, on day 1, 3, 7 and 14 (before necropsy) after administration.
- Necropsy
On day 14 after the administration, all surviving animals were anesthetized with CO2 gas and terminated by exsanguination from the abdominal aorta and caudal vena cava. Complete post-mortem examinations were performed on all vital organs.
Statistics:
Body weights of all animals in each step were analysed through tables and figures that were applied to the mean value and the standard deviations. Additional
statistical assessment was not performed in data analysis. The LD50 value was classified according to the study procedure diagram of the National Institute of
Environment Research [Notice No. 2010-29] (16th Aug., 2010)
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
1st step 300 mg/kg dosing group:
No mortalities

2nd step 300 mg/kg dosing group:
No mortalities

3rd step 2,000 mg/kg dosing group:
All animal died on day 0 and day 1 after administration. No. 8 animal had crawling position and died at 30 minutes after administration. No. 9 animal showed crawling position at 30 minutes, coma and terminal respiration at 1 hour and finally died at 2 hours after administration. In No. 7 animal, crawling position also was observed at 30 minutes after administration. Crouching position and piloerection were observed at 1 hour after administration, and then piloerection only persisted. The animal was found dead on day 1.
Clinical signs:
1st step 300 mg/kg dosing group:
No unusual clinical signs were observed during the observation period.

2nd step 300 mg/kg dosing group:
No unusual clinical signs were observed during the observation period.

3rd step 2,000 mg/kg dosing group:
All animal died on day 0 and day 1 after administration. No. 8 animal had crawling position before death. No. 9 animal showed crawling position at 30 minutes, coma and terminal respiration at 1 hour. In No. 7 animal, crawling position also was observed at 30 minutes after administration. Crouching position and piloerection were observed at 1 hour after administration, and then piloerection only persisted.
Body weight:
1st step 300 mg/kg dosing group:
In body weights, body weight on day 7 of one animal (No. 1) was decreased compared with that on day 3. Except that, normal body weight gains were detected.

2nd step 300 mg/kg dosing group:
There were normal body weight gains in all animals.

3rd step 2,000 mg/kg dosing group:
Not applicable as all animals died on day 0 and day 1 after administration.
Gross pathology:
1st step 300 mg/kg dosing group:
In necropsy at the termination of observations, no abnormal gross findings were observed.

2nd step 300 mg/kg dosing group:
In necropsy at the termination of observations, no abnormal gross findings were observed.

3rd step 2,000 mg/kg dosing group:
From the results of necropsy of the decedents, gross findings were observed as follows; In two animals died on day 0, glandular stomach was coated with black material and area of liver where is adjacent to stomach was discolored with gray-green (No. 8 and 9). In an animal died on day 1, gastric filling with black substance and black coating in glandular stomach were founded in stomach and area of liver where is adjacent to stomach was discolored with light brown (No. 7).
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The test substance ESA is considered to be classified as GHS category 4 (300 < LD50 cut-off ≤ 2,000 mg/kg b.w.) under present study conditions.
Executive summary:

The test substance, ESA, was administered in single dose to female Sprague-Dawley (SD) to evaluate the lethal dose 50 (LD50) and acute toxicity. A starting dose level was set as 300 ㎎/㎏ because the toxicological information of the test substance was not known. During study period, mortalities, clinical signs and body weight changes and gross findings at necropsy were examined.

In the first and second steps (300 ㎎/㎏), No mortalities and unusual clinical signs were observed during the observation period. In body weights, there were no changes related with the test substance. And abnormal gross findings were not detected in necropsy at the termination of observations.

In the third step (2,000 ㎎/㎏), all animals died on the day of dosing (day 0) and day 1. Crawling position was observed in all animals at 30 minutes after administration.

No. 8 animal died immediately. No. 9 animal showed coma and terminal respiration at 1 hour and died at 2 hours after administration. No. 7 animal had crouching position and piloerection at 1 hour after administration and piloerection persisted on day 0. The animal died on day 1 finally. Necropsy of the decedents revealed the following; In animals died on day 0, glandular stomach was coated with black material and area of liver where is adjacent to stomach was discolored with gray-green. In an animal died on day 1, gastric filling with black substance and black coating in glandular stomach were founded in stomach and area of liver where is adjacent to stomach was discolored with light brown.

Therefore, the test substance ESA is considered to be classified as GHS category 4 (300 < LD50 cut-off ≤ 2,000 ㎎/㎏ b.w.) under present study conditions.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw
Quality of whole database:
The available data is sufficient to evaluate this endpoint.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Acute Oral Toxicity:

In the study the test substance was dissolved in sterilized distilled water (when it was heated in boiling water at 40°C) to prepare the test solutions. Based on the known hydrolysis of the test substance the stability of the test substance has been considered for this study.

The LD50 value of acute oral toxicity of the test substance determined in vivo was also assessed with means of computational tools in order to support the validity of the experimental study, which could be affected by the rapid hydrolysis of the test item.

The performed QSAR modelling with ESA showed that the acute oral toxicity LD50 in rat ranges from 179 to 844 mg/kg bw. These data, except the low reliable US EPA TEST prediction (LD50: 179 mg/kg) are in line with the experimentally observed LD50 dose range.

Model

Prediction

Reliability

Acute Rat Oral Toxicity LD50

 

 

US EPA TEST

- Consensus: Hierarchical clustering / FDA / Nearest neighbour

179 mg/kg

 

low

Danish QSAR Database

- Rat Oral (ACD Labs)

300 mg/kg

borderline = low

TOPKAT

- Rat LD50 (extensible 76 comp)

844 mg/kg

mod

It may be concluded that the experimentally obtained LD50 value for ESA and the estimated LD50 values with computational tools were approximatively in the same toxicity range and reliable for GHS category 4 classification.

Justification for classification or non-classification

The substance has an LD 50 value of >300 <= 2000 mg/kg bw via the oral route and according to CLP is classified as acutely toxic, Category 4.