Potassium 2-ethylhexanoate

Endpoint summary

Description of key information

Skin irritation: irritating (OECD 404, GLP)
Eye irritation: serious eye damage (OECD 405; GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-21 to 2013-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: approximately 6 - 7 months
- Housing: before and after the 4-hour exposure period, the animals were kept singly in cages measuring 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schönwalde, Germany). During the exposure period, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn. The cages excluded irritation of the skin by excrements and urine.
- Diet (ad libitum; before and after exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum; before and after exposure period): drinking water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg of the test item was applied to the test site.
1000 mg of the test item were mixed with 500 mg aqua ad iniectabilia (Batch no. 123868001, B. Braun Melsungen AG, 34212 Melsungen, Germany , 750 mg of this paste were applied per animal (approx. 500 mg test item/animal)

Duration of treatment / exposure:

4 hours

Observation period:

Prior to the administration and 60 minutes, 24, 48 and 72 hours after the exposure period

Number of animals:

3 female rabbits

Details on study design:

TEST SITE
- Area of exposure: approximately 24 hours before the test, the fur was removed by closely clipping the dorsal area of the trunk of the animals. Care was taken to avoid abrading the skin. Only animals with healthy intact skin were used.
The test item was applied to the test site (area: approx. 6 cm2) and then covered with a gauze patch. The patch was held in contact with the skin with non-irritating tape for the duration of the exposure period. The surrounding untreated skin served as a control.

INITIAL TEST AND CONFIRMATORY TEST
As it was expected that the test item would not produce any irritancy, all 3 animals were applied at one time, i.e. no initial test was carried out.

SCORING SYSTEM: according to the Draize scale

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 12 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

Category 2 (irritant)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is irritating to the skin.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is irritating to the skin (R 38).
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is classified for skin irritation cat. 2 (H315).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2009-09-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Prior to the application a 20% (w/v) solution of the test item (793.45 mg) in the vehicle (3.96 mL) was prepared.

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

NOTE: Due to equivocal results three experiments needed to be performed.

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded.

OUTLINE OF STUDY
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 181012)) or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 181012). The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item. Sets of three corneae were used for treatment with the test items and the negative and positive controls.
The anterior compartment received the test item solution or negative or positive control at a volume of 0.75 mL each on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath in a horizontal position for 240 minutes.
After the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water, fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

HISTOPATHOLOGY (only corneae of experiment 3)
After final opacity and permeability measurement, the corneae exposed to the test item and to the controls were transferred to vials containing a fixative (32.35% (v/v) deionised water, 32.35% (v/v) methanol, 24.5% (v/v) formaldehyde, and 10.8% (v/v) glacial acetic acid) and were sent to a laboratory for slide preparation and histopathological investigation. The corneae were processed and stained by Haematoxylin and Eosin according to the laboratory SOPs.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea. The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Irritation parameter:

other: in vitro irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

55.57

Remarks on result:

other: Experiment 1

Irritation parameter:

other: in vitro irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

35.59

Remarks on result:

other: Experiment 2

Irritation parameter:

other: in vitro irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

26.36

Remarks on result:

other: Experiment 3

Irritant / corrosive response data:

Due to equivocal results three experiments needed to be performed.
Relative to the negative control, the test item potassium 2-ethylhexanoate caused an increase of the corneal opacity and of the permeability in the 1st experiment. The calculated mean in vitro irritation score was 55.57. According to OECD 437 the test item is formally classified as corrosive / severe irritant to the eye. Nevertheless since one of the treated corneas did show no corrosive effect (irritation score 45.16), one showed a borderline corrosive effect (irritation score 56.08), and one showed a clear corrosive effect (irritation score 64.47) the result has to be considered as equivocal. In the 2nd experiment also an increase of the corneal opacity and of the permeability could be observed. The calculated mean in vitro irritation score was 35.59 indicating no corrosive effect of the test item. Nevertheless also the result of the 2nd experiment has to be considered as equivocal since two corneas showed no corrosive effect (irritation scores of 1.03 and 10.99) and one cornea showed a strong effect (irritation score 94.75). In the 3rd experiment none of the test item treated corneae did show a corrosive effect (irritation scores 26.04, 51.24, and 1.79) indicating no corrosive effect of the test item.

Other effects:

Histopathology (please refer to table 4 in the field "Any other information on results incl. tables" below):
For the 3rd experiment the corneae were evaluated histopathologically in order to obtain more information on the irritating effect of the test item.
The results obtained indicate an effect of the test item on the corneal sample, while no effects could be observed on the negative control corneae. Whilst the positive control sections showed a diffuse or localized erosion of the corneal epithelium; the change was confined to the superficial layers of cells. In those animals treated with potassium 2 -ethylhexanoate the changes were focal (or multifocal in sample 7) but deeper into the epithelium. Sample 8 was not able to be assessed due to damage to the tissue where the epithelium was missing. This is considered to be artefactual.
Application of potassium 2 -ethylhexanoate to the bovine corneal samples produce a focal or multifocal degeneration and erosion of the epithelial cells.

Table 1: Results of the 1st Experiment after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Irritation Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	1.00	0.109	0.086	2.64	2.29	Non corrosive / non severe irritant
	1		0.078		2.17
	1		0.070		2.05
Positive Control	155.00*		- 0.023*		154.66	181.55	Corrosive / severe irritant
	191.00*		- 0.036*		190.47
	200.00*		- 0.032*		199.53
Potassium 2-ethyl- hexanoate	56.00*		0.631*		65.47	55.57	Corrosive / severe irritant
	39.00*		0.410*		45.16
	44.00*		0.805*		56.08

*corrected values

Table 2: Results of the 2nd Experiment after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Irritation Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	1	1.00	0.048	0.049	1.72	1.74	Non corrosive / non severe irritant
	1		0.044		1.66
	1		0.056		1.84
Positive Control	215.00*		0.007*		215.10	212.55	Corrosive / severe irritant
	213.00*		0.013*		213.19
	209.00*		0.024*		209.36
Potassium 2-ethyl- hexanoate	1.00*		0.002*		1.03	35.59	Non corrosive / non severe irritant
	10.00*		0.066*		10.99
	90.00*		0.317*		94.75

*corrected values

Table 3: Results of the 3rd Experiment after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Irritation Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	5	2.00	0.052	0.055	5.78	2.83	Non corrosive / non severe irritant
	1		0.055		1.83
	0		0.059		0.89
Positive Control	146.00		0.001		146.01	213.03	Corrosive / severe irritant
	316.00		0.002		316.03
	177.00		0.003		177.04
Potassium 2-ethyl- hexanoate	14.00		0.803		26.04	26.36	Non corrosive / non severe irritant
	39.00		0.816		51.24
	1.00		0.053		1.79

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed in all three experiments (irritation scores 2.29, 1.74, and 2.83).

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacityon the corneae corresponding to a classification as corrosive / severe irritant to the eye (CLP/EPA/GHS (Cat 1)) in all three experiments (irritation scores 181.55, 212.55, and 213.03).

Table 4: Histopathological Evaluation

Dose Group	Cornea No.	Observations
Negative Control	1	No abnormality detected
	2	No abnormality detected
	3	No abnormality detected
Positive Control	4	Diffuse epithelial erosion, grade 2
	5	Localised epithelial erosion, grade 1
	6	Diffuse epithelial erosion, grade 1
Test Item Cornea	7	Multifocal epithelial degeneration/erosion, grade 3
	8	No epithelial evaluation possible due to damage, further section no better
	9	Focal epithelial degeneration/erosion, grade 1

Table 5: Historical data

	Positive control	Negative control
Mean in vitro Irritation Score	176.71	1.78
Standard Deviation	42.65	0.75
Range of in vitro irritation scores	99.4 - 292.3	0.41 - 2.99

Values of 138 studies with solid test items performed until November 2012

Interpretation of results:

other: no clear conclusion can be drawn from the results, since the results are equivocal.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

It is not possible to draw a conclusion about the corrosive potential of this test substance from the results obtained in this study.

Relative to the negative control, the test item potassium 2-ethylhexanoate caused an increase of the corneal opacity and of the permeability in the 1st experiment. The calculated mean in vitro irritation score was 55.57. According to OECD 437 the test item is formally classified as corrosive / severe irritant to the eye. Nevertheless since one of the treated corneas did show no corrosive effect (irritation score 45.16), one showed a borderline corrosive effect (irritation score 56.08), and one showed a clear corrosive effect (irritation score 64.47) the result has to be considered as equivocal. In the 2nd experiment also an increase of the corneal opacity and of the permeability could be observed. The calculated mean in vitro irritation score was 35.59 indicating no corrosive effect of the test item. Nevertheless also the result of the 2nd experiment has to be considered as equivocal since two corneas showed no corrosive effect (irritation scores of 1.03 and 10.99) and one cornea showed a strong effect (irritation score 94.75). In the 3rd experiment none of the test item treated corneae did show a corrosive effect (irritation scores 26.04, 51.24, and 1.79) indicating no corrosive effect of the test item. In the histopathological evaluation no effects could be observed on the negative control corneae and distinct effects were observed on the positive control corneae. For the test item treated corneae one cornea showed strong erosion (grade 3), one cornea showed strong damage which made the evaluation of erosive effects impossible, and the third cornea showed erosive effects (grade 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation

One reliable in vivo study described by Hansen (2013)(OECD 404; GLP compliant) is considered to be reliable without restrictions.The substance was determined to be irritating to the skin.

Furthermore, a reliable in vitro study described by Heppenheimer (2013)(OECD 435; GLP compiant) is considered to be reliable without restrictions. The substance was determined not to be corrosive to the skin.

Eye irritation

One reliable in vitro study described by Heppenheimer (2013) (OECD 437; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be corrosive or severely irritating to the eye.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Skin irritation

Reference Hansen (2013) is considered as the key study for in vivo skin irritation and will be used for classification. The skin irritation was scored according to the Draize scale. The mean score (24, 48, 72 h) for erythema and oedema for all three animals were as follows:

Erythema: 1, 3, 2 (not fully reversible within 14 days)

Oedema: 0 for all animals

Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will be classified as skin irritating cat. 2 (H315). Furthermore, the substance is classified as skin irritant (R38) according to Directive 67/548 EC.

Eye irritation

Reference Heppenheimer (2013) is considered as the key study for severe eye irritation and will be used for classification. Histopathological investigations of the corneae shows focal or multifocal degeneration and erosion of the epithelial cells.

The classification criteria according to regulation (EC) 1272/2008 as severe eye irritation are met, hence the substance is classified as Category 1 (H318). Furthermore, the substance is classified as risk of serious damage to eyes (R41) according to Directive 67/548 EC.