butanedioic acid, 2-octenyl

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

butanedioic acid, 2-octenyl

EC Number:

820-780-3

Cas Number:

62568-82-5

Molecular formula:

C12 H20 O4

IUPAC Name:

butanedioic acid, 2-octenyl

Test material form:

other: White solid

Details on test material:

Test Item Name : Octenylsuccinic Acid
Appearance/Colour/Odour : White solid
Lot N° : 8515025

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animals:
Species : Rat (Rattus norvegicus)
Strain : Wistar (RCCHan:WIST)
Age/Weight at dosing : 8 to 12 weeks, Weight (g) Minimum: 166.3, Maximum: 202.2
Source : Animal Breeding Facility, Jai Research Foundation
Total Number of Animals Used : Eight females (nulliparous and non-pregnant)

Acclimatisation:
Acclimatisation Period : 6 to 16 days
The rats were received into the experimental procedure room and allowed to acclimatise for a period of 6 to 16 days prior to commencement of dosing.

Husbandry Practices:
Caging : Rats were housed individually in standard polypropylene solid bottom cages which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). These cages have stainless steel top grills with steam sterilized corn cob bedding material and facilities for pelleted feed and drinking water (polypropylene bottle fitted with sipper tube). Rack units were rotated once in a week.
Water Bottle : Each cage was supplied with a polypropylene water bottle with a stainless steel nozzle.
Enrichment : Rats were provided with a wooden block in each cage.
Room Sanitation : Daily: 1. Rack was cleaned with cloth, 2. Floor of experimental procedure room was swept, 3. All work tops and the floor were mopped with a disinfectant solution.

This study complied with all applicable sections of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines and Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011).

Animal Identification:
Each rat was uniquely numbered on the tail using a tattoo machine. Appropriate labels were attached to the cages indicating the study number, test item code and sex, dose, study code, cage number and animal number.

Feed and Water:
The rats were provided with feed and water, ad libitum (with the exception of overnight fasting and three hours post-dosing). The quality of feed and water is regularly monitored at Jai Research Foundation; copies of the relevant certificates of analysis are kept in the study file. There were no known contaminants in the feed and water at levels that would have interfered with the experimental results obtained.

Environmental Conditions:
Animal Room : BMR 27, Department of Toxicology
Environmental Conditions:
Temperature : 20 to 23 °C
Relative Humidity : 49 to 58%
Air Changes : 17 air changes/hour
Photoperiod : The photoperiod was 12 hours artificial light and 12 hours darkness, light hours being 06:00 h – 18:00 h which was maintained through an automatic timer.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dose Administration:
The test item was mixed with reverse osmosis water to a concentration of 200 (for rat Nº 1), 17.5 (for rat Nº 2), 55 (for rat Nº 3, 5 and 7) and 175 (for rat Nº 4, 6 and 8) mg/mL and then adjusted according to the body weight to permit a constant dose volume (10 mL/kg body weight). Individual dose volumes were adjusted according to body weight and dose level. All rats were dosed by oral gavage (day 1) using a metal cannula attached to a BD 1 mL disposable syringe which was graduated up to 1 mL. Rats were fasted overnight prior to dosing and until three hours post-dosing.

Doses:

Initially limit test was conducted with the dose level of 2000 mg/kg body weight in rat N° 1. This rat was found dead on day 2 post dose. Hence, a Main Test was conducted with the starting dose level of 175 mg/kg body weight in rat N° 2 (slope 2). The first rat survived; hence the six additional female Wistar rats received doses of 550 (rat N° 3, 5 and 7) and 1750 (rat N° 4, 6 and 8) mg/kg body weight according to the Up and Down Procedure. Stopping criteria for stop dosing for this test met: 5 reversals in 6 tests.

No. of animals per sex per dose:

Eight animals total tested. Initially limit test was conducted with the dose level of 2000 mg/kg body weight in rat N° 1. This rat was found dead on day 2 post dose. Hence, a Main Test was conducted with the starting dose level of 175 mg/kg body weight in rat N° 2 (slope 2). The first rat survived; hence the six additional female Wistar rats received doses of 550 (rat N° 3, 5 and 7) and 1750 (rat N° 4, 6 and 8) mg/kg body weight according to the Up and Down Procedure. Stopping criteria for stop dosing for this test met: 5 reversals in 6 tests.

Control animals:

no

Details on study design:

Observations:
The rats were observed for signs of toxicity and mortality at ½, 1, 2, 3, 4 and 6 hours postadministration on the day of dosing. Subsequently, the rats were observed twice a day for morbidity and mortality for a period of 14 days following oral dosing. The clinical signs were recorded once a day. Individual body weight was recorded prior to dosing on day 1, and on days 8 and 15.

Necropsy:
At study termination, all surviving rats were humanely euthanised by carbon dioxide asphyxiation. All rats on the study were subject to a gross pathological examination consisting of an external examination and opening of abdominal and thoracic cavities. The stomach of each rat was opened, the
contents rinsed/removed, and the mucosal surface was examined for signs of irritation, erosions, ulcers or any other findings. Gross macroscopic changes, if any, were recorded.

Statistics:

The LD50 was calculated using the Dixon’s maximum likelihood method using software (AOT 425 StatPgm).

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

1 030 mg/kg bw

Based on:

test mat.

95% CL:

>= 550 - <= 1 750

Mortality:

Mortalities:
Rats treated at 175 (1 rat, rat N° 2) and 550 (3 rats, rat N° 3, 5 and 7) mg Octenylsuccinic Acid/kg body weight survived through the 14-day observation period. Rats treated at 2000 (1 rat, rat N° 1) and 1750 (3 rats, rat N° 4, 6 and 8) mg Octenylsuccinic Acid/kg body weight died by day 2 post dosing.

Clinical signs:

Clinical Observations:
No signs of toxicity were observed in the rats treated with 175 (rat N° 2) and 550 (rat N° 3, 5 and 7) mg/kg body weight. Lethargy was observed in the rats treated with 1750 (rat N° 4, 6 and 8) mg/kg body weight whereas lethargy and piloerection were observed in rat N° 1 treated with 2000 mg/kg
body weight.
All surviving animals were active and healthy during the study.

Body weight:

Body Weights:
There was a normal increase in the body weight of all the surviving rats during the experimental period

Gross pathology:

Terminal Studies (Macroscopic Findings)
External:
External examination of the found dead and terminally sacrificed rats did not reveal any abnormality.
Internal:
Internal examination of found dead rats revealed lesions such as congestion of the liver (rat N° 1, 4, 6 and 8) and congestion of the lungs (rat N° 1, 4 and 8), whereas the terminally sacrificed rats did not reveal any lesions. Lesions observed in the found dead rats could be correlated with the test item used in the present study.

Applicant's summary and conclusion

Interpretation of results:

Toxicity Category IV

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute oral estimated LD50 of the Octenylsuccinic Acid was estimated to be 1030 mg/kg body weight (Based on an assumed sigma of 0.5) in female Wistar rats with approximate 95% confidence interval is 550 to 1750 mg/kg body weight.

Executive summary:

An acute oral toxicity study (Up-and-Down Procedure) was conducted using eight female Wistar rats given a single oral dose of Octenylsuccinic Acid in reverse osmosis water. Initially a limit test was conducted with the dose level of 2000 mg/kg body weight in rat N° 1. This rat was found dead on day 2 post dose. Hence, a Main Test was conducted with the starting dose level of 175 mg/kg body weight in rat N° 2. This rat survived; hence the six additional female Wistar rats received doses of 550 (rat N° 3, 5 and 7) and 1750 (rat N° 4, 6 and 8) mg/kg body weight according to the Up and Down Procedure.

No signs of toxicity were observed in the rats treated with 175 (rat N° 2) and 550 (rat N° 3, 5 and 7) mg/kg body weight. Lethargy was observed in the rats treated with 1750 (rat N° 4, 6 and 8) mg/kg body weight whereas lethargy and piloerection were observed in rat N° 1 treated with 2000 mg/kg body weight. Rat N° 1, 4, 6 and 8 were found dead on day 2.

All surviving rats were active and healthy during the study. All surviving rats gained body weight by the end of the study.

External and internal examination of terminally sacrificed rats did not reveal any lesions of pathological significance. Internal examination of found dead rats revealed congestion of the liver (rat N° 1, 4, 6 and 8) and congestion of the lungs (rat N° 1, 4 and 8). Lesions observed in found dead rats could be correlated with the test item used in the present study.

The acute oral estimated LD50 of the Octenylsuccinic Acid was estimated to be 1030 mg/kg body weight (Based on an assumed sigma of 0.5) in female Wistar rats with approximate 95% confidence interval is 550 to 1750 mg/kg body weight.