Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: Colorless clear liquid
- Storage Conditions: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
Experimental Animals
Eleven (11) females Sprague-Dawley strain SPF rats [Crl:CD(SD), Atsugi Breeding Center, Charles River Laboratories Japan, Inc.], were purchased at 7 weeks of age, and quarantined / acclimated to the test environment for over a week at the test facility. Healthy animals were selected after the quarantine/ acclimation period on the basis of the clinical signs and body weight and used in the study at 8 weeks of age. Individual body weight at the time of administration was in the range of 187 to 199 g. Group allocation was conducted 1 or 2 days before administration of each dose step. All animals to be used for group allocation were weighed, and animals were selected in such a way that the body weight of animals for the dose step group and group mean body weight between each dose step was comparable as far as possible. The animals were individually identified by marking with ear tags for small animals at the time of receipt. Labels, color-coded according to administration date, indicating the study number, administration route, dose level, sex, animal number, ear tag number and date of administration were attached to the cages. Animals remaining were excluded from the study on
day 5 after administration of the third dose step and were transferred to the animal care manager for effective utilization.
Note: The number of animals ordered according to the Protocol was 10 females, but the actual number of animals received was 11 females.

Animal Husbandry
The animals were housed in an animal room (Room Number: 701) which was controlled to maintain the temperature at 23 +/- 3°C, the relative humidity at 50 +/- 20%, the air ventilation at 10 to 15 times per hour and 12-hour lighting per day (07:00 to 19:00). The animals were housed
individually in bracket-type stainless-steel wire mesh cages (W 254 x D 350 x H 170 mm:Lead Engineering Co., Ltd.). Animals were allowed free access to pelleted diet CR-LPF (Miation-sterilized Oriental Yeast Co., Ltd., Lot Nos.: 130411, 130517) and tap water (Gotemba City Water, using water bottles). During the period of animal experiment, the actual temperature of the animal room was between 22 and 24°C, and the relative humidity was
between 39 and 75% . Analysis of contaminants in the feed was carried out for the lot by Eurofins Scientific Analytics and analysis of water contaminants was carried out on a quarterly basis according to the Waterworks Law by Shibaura Semtek Co., Ltd. Copies of all data obtained from the above facilities were filed after verifling that there were no effects on the study results.
Note: Relative humidity was over 70% between 15:50 and 16:30 on August 23, 2013, which deviated from the acceptable range specified by protocol (50 +/- 20%), due to blackout from lightning damage. However, it was a slight change and there were no abnormalities and no effects on the result of study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The formulation received was used as the test substance without modification. Therefore, there was no preparation process.

Oral administration was selected in accordance with the toxicity study guidelines. The test substance was administered using a stomach tube once orally to rats which had been fasted overnight (approximately 18 hours) fiom the previous day. Individual dose volume was calculated on the basis of body weight on the day of administration (day 0 of administration).
Animals were allowed free access to feed again after the end of clinical observation at 4 hours after dosing.
The animals were observed for 14 days after administration.
Doses:
Since the acute oral toxicity of the test substance was expected to be low, the starting dose level was set at 2000 mg/kg . For the subsequent dose, the dose level for the second and third dose steps was set at 300 mg/kg in accordance with the test procedure in the toxicity study guideline "Acute Toxic Class Method".
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
Method of Observation, Measurement and Examination
The day of administration was counted as day 0 of administration and the following observation and measurement were conducted.

Observation of Clinical Condition
The animals were observed frequently for the first 6 hours after administration (from immediately after administration to 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 6 hours after administration), and once daily (between 8:43 and 10:28) thereafter for 14 days, for clinical signs such as abnormalities of external appearance, nutritional condition, posture, behavior and excretions.

Measurement of Body Weight
Body weight was measured on the day of administration (immediately before dosing) and on days 1,3,7 and 14 after administration (between 8:50 and 10:27).

Pathological Examination
Soon after discovery of the animals that died, and after survivors were sacrificed by exsanguinations (via the abdominal aorta) under isoflurane anesthesia at the end of the 14-day observation period, external appearance and organs/tissues in the cranial, thoracic and abdominal cavities were examined macroscopically. Organs/ tissues were not preserved since necropsy revealed no abnormalities.

Statistics:
Based on the death occurrence during the 14-day period after administration, approximate LD50 value was estimated.

For body weight, mean with standard deviation was calculated for each measurement day for each administration stage. In addition, body weight gain during the observation period from day 0 of administration to day 14 after administration was determined and mean with standard
deviation calculated in the same manner.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals died in the first dose step at 2000 mg/kg: however, no deaths occurred in the second and third dose steps at 300 mg/kg. Thus, it was estimated that the LD50 was higher than 300 mg/kg but lower than 2000 mg/kg.
Clinical signs:
In the dose step at 2000 mg/kg , 1 animal died at 6 hours after administration and 2 animals died on day 1 after administration. The 2 animals that died on day 1 after administration showed decreased spontaneous movement and reddish urine at 6 hours after administration.
In the both dose steps at 300 mg/kg, all animals showed reddish urine from 6 hours after administration, but it disappeared by day 2 after administration and there were no abnormalities thereafter.
Body weight:
In both dose steps at 300 mg/kg, body weight increase was slightly low on day 1 after administration and suppressed body weight gain was observed, but body weight increase was normal thereafter.
Gross pathology:
In the animals that died in the dose step at 2000 mg/kg and in the surviving animals in both dose steps at 300 mg/kg, there were no abnormalities in the external appearance or organs/tissues in the cervical, thoracic or abdominal cavities in any animal.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Remarks:
Migrated information
Conclusions:
On the basis of the results described above, it was estimated that the LD50 value of Isopropoxyethyl salicylate was higher than 300 mg/kg bw but lower than 2000 mg/kg bw when administered orally to rats once.
Executive summary:

The acute oral toxicity of Isopropoxyethyl salicylate was examined according to OECD guideline 423 and GLP principles in Sprague-Dawley rats (3 females per dose step). All animals died at 2000 mg/kg bw, however, no deaths occurred at 300 mg/kg bw. All animals exposed to 2000 mg/kg died 6 hours after administration or on day 1 after administration. The animals that died on day 1 after administration showed decreased spontaneous movement and reddish urine at 6 hours after administration. All animals in the 300 mg/kg bw showed reddish urine starting 6 hours after administration, but this effect disappeared by day 2 after administration and there were no abnormalities thereafter. At 300 mg/kg bw, body weight increase was slightly low on day 1 after administration and suppressed body weight gain was observed, but body weight increase was normal thereafter. There were no abnormalities in the results of necropsy of the animals that died or that survived. On the basis of the results described above, it was estimated that the oral LD50 value of Isopropoxyethyl salicylate was higher than 300 mg/kg but lower than 2000 mg/kg bw.

Based on these data, the LD50 for Isopropoxyethyl salicylate was determined to be between 300 and 2000 mg/kg bw, the substance is classified for oral toxicity in category 4 according to Regulation (EC) 1272/2008.