Calcium bis(2-ethylhexanoate)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2021 - 05 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Calcium bis(2-ethylhexanoate)

EC Number:

205-249-0

EC Name:

Calcium bis(2-ethylhexanoate)

Cas Number:

136-51-6

Molecular formula:

C8H16O2.1/2Ca

IUPAC Name:

calcium bis(2-ethylhexanoate)

Test material form:

solid: particulate/powder

Remarks:

off white powder lumping

Details on test material:

Supplier: ORGANOMETAL S.A.
CAS Number: 136-51-6
Batch/Lot number: 2012291021
Appearance: off white powder lumping
Purity: 98% + 2% water
Ca content: 12 %
Expiry date: 29 October 2022

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST rats

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
- Hygienic level at supplier: SPF
- Hygienic level during the study: Standard housing conditions
- Number of animals: 12 animals, 3 animals/group
- Sex: Female, nulliparous and non-pregnant animals
- Age of animals at dosing: Young adult rats, approx. 8-10 weeks old
- Body weight range at dosing: 169-206 g. The maximum difference of individual animal weights from the mean of the treatment group did not exceed 20%.
- Acclimatisation period: At least 8 days
- Animal health: Only healthy animals were used for the test. The health status was certified by the Veterinarian.
- Housing: Group caging (3 animals/cage)
- Cage type: T3H polycarbonate
- Bedding and nesting: “SAFE 3/4-S-FASERN” certified wooden chips and “Sizzle pet” nest material were available to animals during the study.
- Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
- Animals received standard laboratory rat diet, ad libitum, and tap water from the municipal supply, as for human consumption from drinking bottles designed for rodents, ad libitum.
- The night before treatment, the animals were fasted. Food, but not water, was withheld overnight. Animals were weighed before dosing. Food was replaced 3 hours after the treatment.

ENVIRONMENTAL CONDITIONS
- Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 20 – 23 °C (target: 22 ± 3 °C)
- Relative humidity: 36 – 58 % (target: 30 – 70 %)
- Ventilation: 15-20 air exchanges/hour

IN-LIFE DATES: From 16 December 2021 to 05 January 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1% methyl cellulose in distilled water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 or 30 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Justification for choice of vehicle: The test item did not dissolve in distilled water, but in 1% methyl cellulose solution formed a suspension which was stable while stirring and was thus chosen as vehicle for this study. Details of the vehicle used in the study were as follows:
Component 1
Name: Methylcellulosum
Batch number: 8075546
Manufacturer: Magilab Kft.
Expiry date: 10 July 2022
Component 2
Name: Aqua purificata (Distilled water)
Batch number: 2108-5518
Manufacturer: Parma Produkt Kft.
Expiry date: 18 February 2022

DOSAGE PREPARATION: The test item was freshly formulated in the vehicle at the appropriate concentration (200 or 30 mg/mL), in the Pharmacy of NEXTREAT Laboratories on the day of administration. The formulations were stirred with magnetic stirrer up to finishing the treatment.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: As starting dose level for acute toxicity study, a dose of 2000 mg/kg body weight (bw) has been selected based on the data, provided by the Supplier, which indicate that the LD50 value of the test item is above 2000 mg/kg bw.
- Initially three animals were treated at the starting dose of 2000 mg/kg bw (Group 1). Two of the three animals survived, one was found dead on Day 1. Therefore, a second group of three animals (Group 2) was treated at the same dose level (2000 mg/kg bw). In the second group, one animal was pre-terminally euthanized due to animal welfare reasons. The other two animals were found dead on Day 1. Based on these results, another group of three animals was treated with a dose of 300 mg/kg bw (Group 3). All animals of Group 3 survived and thus a further group of three animals (Group 4) was treated with 300 mg/kg bw. As no animals died in this confirmatory group, no further testing was required according to the criteria for termination given in Annex 2d of OECD Guideline No. 423.

Doses:

2000 mg/kg bw or 300 mg/kg bw

No. of animals per sex per dose:

12 (3 animals per group)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Clinical observations: Following the end of the dosage, the animals were observed individually once during the first 30 minutes, at 1, 2, 3, 4 and 6 hours after the treatment and once daily for 14 consecutive days thereafter. Individual observations were performed on the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The time of death was recorded as precisely as possible.
- Body weight: The body weight of the animals was recorded on Days 0 (prior to dosing), 7 and 14 (prior to necropsy), with a precision of 1 g. Terminal body weight of animals found dead was also recorded.
- Necropsy: Animals were subjected to a necropsy and a macroscopic examination. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross macroscopic changes were recorded for each animal.

Statistics:

The method used was not intended to allow the calculation of a precise LD50 value.

Results and discussion

Effect levels

Effect levels 1

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - <= 2 000 mg/kg bw

Based on:

test mat.

Effect levels 2

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

At the dose level of 2000 mg/kg bw, 1/6 animals was euthanized pre-terminally because of animal welfare reasons on Day 0. Three animals (out of 6) were found dead on Day 1, two animals survived.
At the dose level of 300 mg/kg bw, no mortality was observed.

Clinical signs:

other:

Body weight:

lower than 10% body weight loss

Remarks:

There was no evidence of effects on body weight or body weight gain that could be attributed to treatment with the test item at any of the doses.

Gross pathology:

A single oral gavage of the test item to female Han:WIST rats, at dose level of 2000 mg/kg bw has led to death 3 out of six animals. Two of these animals showed dark red discoloration of the glandular mucosa of the stomach and of the mucosa of the duodenum and jejunum. One showed thickening of the non-glandular region of the stomach and pale, multifocal discoloration of the liver in all lobes.
In the one pre-terminally euthanized animal, the test item was found as a white, solid, gypsum-like clump in the stomach, entirely filling it. The formation of this a clump is most likely a result of the interaction of the test item with the divalent cations contained in the drinking water (reproduced, post-hoc, in vitro), and thus was a result of
the drinking activity of this animal immediately after the application of the formulated test item. In case of this animal, this physicochemical behavior (flocculation/precipitation) of the test item is the likely cause of death.
Other findings such as gas in the stomach or collapsed lungs in the animals found dead on Day 1 are not test item-related.
The two surviving animals of this group were without pathological changes at the time of the scheduled necropsy.
In the 300 mg/kg bw group, no macroscopic changes were observed.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Remarks:

The test substance is classified Category 4 according to Regulation (EC) No 1272/2008 (CLP)

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test item Calcium bis(2-ethylhexanoate) was found to be 300 < LD50 ≤ 2000 mg/kg bw in female Han:WIST rats.
The LD50 cut-off value is 2000 mg/kg bw.
The study result triggers the following classification/labelling:
- Regulation (EC) No 1272/2008 (CLP): Category 4
- GHS (rev. 7) 2017: Category 4