2-ethylhexanoic acid, manganese salt

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 404, GLP)
Eye irritation: irritating (OECD 405, GLP)

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:

2013-03-15 to 2013-03-28

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 8 months
- Weight at study initiation: 2.6 - 2.8 kg
- 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 (^ 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 severe irritancy or corrosion, the test was started using at first only one animal, receiving a single patch for an exposure period of 4 hours.
As neither a corrosive effect nor a severe irritant effect was observed after a four-hour exposure, the test was completed using two additional animals, each with one patch only, for an exposure period of 4 hours.

SCORING SYSTEM: according to the Draize scale

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Irritant / corrosive response data:

None of the three rabbits exposed for 4 hours to 500 mg 2-ethylhexanoic acid, manganese salt/animal showed any skin reactions.

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is not irritating to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as skin irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-03 to 2013-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-11-30

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS - New Zealand White (Hsdlf:NZW) strain rabbit
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.29 or 2.72 kg
- Housing: the animals were individually housed in a suspended cage.
- Diet (ad libitum): 2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (ad libitum): mains drinking water
- Acclimation period: at least five days

The animal was provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Relative humidity: 30 to 70%
- Air exchange: at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A volume of 0.1 mL of the test item, which was found to weigh approximately 93 mg was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes.

Duration of treatment / exposure:

not applicable

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours as well as Days 7 and 14 following treatment

Number of animals or in vitro replicates:

2 male rabbits

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

INITIAL AND CONFIRMATORY TEST
Initially, a single rabbit was treated. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale (please refer to table 2 in the field "Any other information on materials and methods incl. tables" below).
After consideration of the ocular responses produced in the first treated animal, a second animal was treated.

SCORING SYSTEM: according to the Draize scale
Any other ocular effects were also noted.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Initial pain reaction: practically no initial pain was observed. Area of cornea involved: one quarter (or less) but not zero (observed from 24 hours to day 7); Slight opacity was observed on day 7 of observation.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Slight inflammation of iris was observed at the 1 hour observation. Slight to severe discharge was observed from 1 hour of observation to day 7 of observation.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate conjunctivae redness was observed at the 1 hour and 7 day observation.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate chemosis was observed at the 1 hour and 7 day observation.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Initial pain reaction: slight initial pain was observed. Area of cornea involved: one quarter (or less) but not zero (observed from 24 hours to 72 hours).

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Slight inflammation of iris was observed at the 1 hour and 7 day observation. Slight to moderate discharge was observed from 1 hour of observation to day 7 of observation.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate conjunctivae redness was observed at the 1 hour and slight conjunctivae redness was observed on day 7 of observation.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Moderate chemosis was observed at the 1 hour and slight chemosis was observed on day 7 of observation.

Irritant / corrosive response data:

Scattered or diffuse corneal opacity was noted in both treated eyes at the 24, 48 and 72-Hour observations and in one treated eye at the 7-Day observation.
Iridial inflammation was noted in both treated eyes one hour after treatment, at the 24, 48 and 72-Hour observations and in one treated eye at the 7-Day observation.
Moderate conjunctival irritation was noted in both treated eyes one and 24 hours after treatment. Severe conjunctival irritation was noted in one treated eye at the 48 and 72-Hour observations. Moderate conjunctival irritation was noted in the other treated eye at the 48-Hour observation with minimal conjunctival irritation at the 72-Hour observation. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 7-Day observation.
Both treated eyes appeared normal at the 14-Day observation.

Other effects:

- Body weight: both animals showed expected gain in bodyweight during the study.

Interpretation of results:

Category II

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material is irritating to the eyes.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the substance is classified as Category 2.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

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 the test item was crushed in mortar with a pistil to improve its consistency. A 20% (w/v) suspension of the test item in 0.9% (w/v) NaCl in deionised water was prepared using ultrasonic technique.

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:

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 Lglutamine, 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 (Oring) 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).

OUTLINE OF STUDY
The anterior compartment received the test item or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 240912)) or 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. 240912)) at a volume of 0.75 mL each on the surface of the corneae and was incubated at 32 ± 1 °C in the waterbath in a horizontal position. Since the test item could not be suspended homogeneously, it was taken care that each 0.75 mL of the prepared stock suspension was distributed evenly to the corneae. The test item, positive control and negatice control were tested in triplicate.
The incubation time lasted 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).

OPACITY MEASUREMENT
The basal opacity of all corneae was recorded (t0). Each corneae with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test items and the negative and positive controls. The opacity was measured again after treatment with the test item, positive control and negative control (t240).

PERMEABILITY DETERMINATION
After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

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:

in vitro irritation score

Run / experiment:

mean

Value:

0.06

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Relative to the negative control, the test item 2-ethylhexanoic acid, manganese salt did not caused any increase of the corneal opacity or permeability. The calculated mean in vitro irritation score was 0.06 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD 437 the test item is classified as not corrosive / not severe irritant to the eye.

Table 1: Results 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	0	0.67	0.104	0.100	1.56	2.17	Non corrosive / non severe irritant
	1		0.097		2.46
	1		0.100		2.50
Positive control	142.33*		0.024*		142.69	156.46	Corrosive / severe irritant
	160.33*		-0.014*		160.12
	166.33*		0.016*		166.57
2 -ethyl-hexanoic acid, manganese salt	0.33*		-0.036*		-0.21	0.06	Non corrosive / non severe irritant
	0.33*		-0.008*		0.21
	0.33*		-0.009*		0.19

* corrected values

- 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 (mean in vitro irritation score 2.17).

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) showed clear opacity of the corneae (mean in vitro irritation score 156.46) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Table 2: 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: not corrosive/not severe irritant to the eye

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item 2-ethylhexanoic acid, manganese salt is not corrosive / not severely irritating to the eye (CLP/EPA/GHS (Cat 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 not to be irritating to the skin.

 

Based on the experience with other metal substances (model substances) in in vitro human skin model test systems for skin corrosion and skin irritation testing, it was decided to deviate from the sequential testing strategy as foreseen in the REACH regulation, Annex VII and VIII in conjunction with Article 12(1). The testing strategy for skin corrosion/irritation testing is presented in the report in Attachment I of the CSR and in endpoint summary of IUCLID Section 7.3.

 

Eye irritation

One reliable in vivo study described by Pooles (2013) (OECD 405; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be irritating to the eyes.

 

Furthermore, a reliable in vitro study described by Heppenheimer (2012)(OECD 437; GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be corrosive nor severely irritating to the eyes.

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
Key study

Effects on eye irritation: 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: 0 for all animals

Oedema: 0 for all animals

 

Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will not be classified as irritating to the skin.

 

Eye irritation

Reference Pooles (2013) is considered as the key study for in vivo eye irritation and will be used for classification. During the study the test item was applied to one eye of two animals each and the eye irritation was scored according to the Draize scale. The following mean scores (24, 48 and 72 hours) were obtained for the two animals:

 

cornea: 1 for both animals

iris: 1 for both animals

conjunctival redness: 2 and 1.67

chemosis: 3 and 1.67

 

All effects were reversible within 14 days. Thus, according to Regulation (EC) 1272/2008 and subsequent amendments the substance will be classified as Category 2.