Calcium bis(2-ethylhexanoate)

Endpoint summary

Description of key information

In a GLP conform OECD 423 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.

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:

16 December 2021 - 05 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - <= 2 000 mg/kg bw

Based on:

test mat.

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.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

> 300 - <= 2 000 mg/kg bw

Quality of whole database:

GLP conform standard test

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

Read-across approach

Selected endpoints for the human health hazard assessment are addressed by read-across, using a combination of data on the metal cation and the organic acid anion. This way forward is acceptable, since metal carboxylates are shown to dissociate to the organic anion and the metal cation upon dissolution in aqueous media. No indications of complexation or masking of the metal ion through the organic acid were apparent during the water solubility and dissociation tests (please refer to the water solubility and dissociation in sections 4.8 and 4.21 of IUCLID). Once the individual transformation products of the metal carboxylate become bioavailable (i.e. in the acidic environment in the gastric passage or after phagocytosis by pulmonary macrophages), the “overall” toxicity of the dissociated metal carboxylate can be described by a combination of the toxicity of these transformation products, i.e. the metal cation and carboxylate anion according to an additivity approach.

 

Calcium bis(2-ethylhexanoate) is the calcium salt of 2-ethylhexanoic acid, which readily dissociates to the corresponding divalent calcium cation and monovalent 2-ethylhexanoate anions. The calcium cation and the 2-ethylhexanoate anion are considered to represent the overall toxicity of calcium bis(2-ethylhexanoate) in a manner proportionate to the free acid and the metal (represented by one of its readily soluble salts). 

 

A detailed justification for the read-across approach is added as a separate document in section 13 of IUCLID.

 

Acute toxicity

An acute oral toxicity study is available for calcium bis(2-ethylhexanoate). Acute dermal and inhalation toxicity will be addressed with existing data on the dissociation products as detailed in the table below. Further details on the acute toxicity of the individual constituents within the framework of regulation (EC) 1907/2006 are given below.

 

Table: Summary of acute toxicity data of calcium bis(2 -ethylhexanoate) and the individual constituents.

	calcium ion	2-ethylhexanoic acid (CAS# 149-57-5)	Calcium bis(2 -ethylhexanoate) (CAS# 136-53-8)
Acute oral toxicity	LD50(rat)= 831 mg Ca/kg bw	LD50(rat) = 2,043 mg/kg bw	LD50 = 2,000 mg/kg bw (measured) LD50 = 1,704 mg/kg bw (read-across)
Acute inhalation toxicity	waived	LD0 = 0.11 mg/L air (nominal)	waived, since the substance is used and placed on the market in a non-inhalable form
Acute dermal toxicity	waived	LD50 > 2,000 mg/kg bw	LD50 >2,000 mg/kg bw (read-across)

 

Under the assumption that the constituents of calcium bis(2-ethylhexanoate) show their toxicological profile individually upon dissolution, the acute oral toxicity of calcium bis(2-ethylhexanoate) can be calculated using the equation given in regulation (EC) 1272/2008, Annex I, Section 3.1.3.6.1. The calculated oral LD50 for calcium bis(2-ethylhexanoate) based on read-across from its constituents is 1,704 mg/kg bw. In an experimental acute oral toxicity study with calcium bis(2-ethylhexanoate) according to the OECD 423 guideline, the LD50 cut-off is 2,000 mg/kg (1 mortality at first step with 2,000 mg/kg bw, 3 mortalities at second step with 2,000 mg/kg bw and no mortalities at 300 mg/kg bw). Because the predicted LD50 based on data for the two transformation products is still below the measured acute oral LD50 cut-off value for calcium bis(2-ethylhexanoate), it can be concluded that read-across for the two transformation products together with the additivity approach to predict the (eco)toxicological effects of the target substance is conservative and no synergistic effects are expected between the transformation products.

 

A study for acute toxicity via inhalation was not conducted with calcium bis(2-ethylhexanoate), since it is produced and placed on the market in a form in which no inhalation hazard is anticipated, thus acute toxic effects are not likely to occur during manufacture and handling of that substance.

 

Conduct of an acute dermal toxicity study is unjustified as the physicochemical properties of the calcium cation do not suggest a significant rate of absorption through the skin. Further the LD50 for the constituent 2-ethylhexanoic acid is above 2,000 mg/kg bw, hence does not require a classification for acute dermal toxicity. Based on the above given information calcium bis(2-ethylhexanoate) is not expected to show any acute toxic effects via dermal route (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006)

 

For further information on the toxicity of the individual constituents, please refer to the relevant sections in the IUCLID and CSR.

Justification for classification or non-classification

The experimentally measured oral LD50 for calcium bis(2-ethylhexanoate) was found to be 300 < LD50 ≤ 2000 mg/kg bw in a GLP conform OECD 423 study. The study result triggers the classification as Acute oral toxicity Category 4 according to regulation (EC) 1272/2008. No classification is required for specific target organ toxicity, single exposure (STOT SE).

Conduct of an acute dermal toxicity study is unjustified as the physicochemical properties of the calcium cation do not suggest a significant rate of absorption through the skin. Further the LD50 for the constituent 2-ethylhexanoic acid is above 2000 mg/kg bw, hence does not require a classification for acute dermal toxicity. Based on the above given information calcium bis(2-ethylhexanoate) is not expected to show any acute toxic effects via dermal route (cf. Annex VIII section 8.5 Column 2 of regulation (EC) 1907/2006).