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Administrative data

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

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)
Version / remarks:
adopted 2006-07-19
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICCVAM Minimum Performance Standards: In Vitro Membrane Barrier Test Systems for Skin Corrosion, June 23, 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICCVAM Recommended Performance Standards for in vitro Test Methods for Skin Corrosion (May 2004)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-03-30
Details on test animals or test system and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 μL of the test item were dispensed directly atop the bio-barrier.
Duration of treatment / exposure:
60 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
TEST SYSTEM
- Test kit:
Name: Corrositex™
Supplier: Transia GmbH, 61239 Ober-Mörlen, Germany
Lot No.: CT052112)

- Preparation of the bio-barrier:
One day prior to testing the bio-barrier matrix was prepared. The bio-barrier powder was solved in the bio-barrier diluent and heated for 20 ± 2 minutes at 68 – 70 °C in a water bath under continuous stirring. The temperature did not exceed 70 °C. The mixture was allowed to cool in the turned-off water bath for another 10 minutes. The mixture was then filled into the membrane holders (200 µL per membrane holder). Air bubbles were avoided. The filled membrane holders were sealed with parafilm and were stored at 2 – 8 °C until further use.

EXPERIMENTAL PERFORMEANCE
- Qualify Test: to test whether the test system is suitable for the test item, 150 μL of the test item were applied into the “Qualify Test Vial”. The vial was shaken until the solution appeared homogenous, and incubated for at least 1 minute. Afterwards, the colour change was noted. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.
- Categorisation Test: the test item was categorised according to the pH value method described in the manufacturer’s manual.
A 10% dilution (v/v) of the test item in deionised water was prepared. The pH value of the dilution was determined. Test items with a pH value of < 7 were added to the “Category A Vial” and test item with a pH value of > 7 were added to the “Category B Vial”. The vial was closed, shaken, and the pH value was determined. Test items showing a pH value ≤ 5 in the “Category A Vial” were assigned to category 1 and test items showing a pH value of ≥ 5
were assigned to category 2. Test items showing a pH value of ≥ 9 in “Category B Vial” were assigned to category 1 and test items showing a pH value of ≤ 9 were assigned to category 2. Since the pH value of the test item dilution in the category A vial was 7.5 the test item was assigned to category 2.
- Classification Test: 7 vials containing the CDS were pre-warmed to room temperature. 4 vials were used for quadruplicate measurement of the test item, one vial was used for the positive control (single measurement), and one vial was used for the negative control (single measurement). The one vial was used as colour reference for the CDS.
The prepared bio-barriers were placed atop the CDS vials (not longer than 2 min prior to application) and 500 μL of the test item or controls (positive control: sulfuric acid 95-97% (lot no. K40281631 929, Merck, 64295 Darmstadt, Germany); negative control: citric acid (lot no. 140986235008107, Sigma, 89555 Steinheim, Germany, 10% (w/v) solution in deionised water)), respectively, were applied per bio-barrier for 1 hour or 4 hours, depending on the results of the categorisation test. The time interval of the possible colour change or precipitation in the CDS solution was recorded.

INTERPRETATION OF THE RESULTS
For each vial the time until observable change in CDS solution was determined. The mean of the quadruplicate measurement the mean was calculated. The test item was categorised according to the following table as can be seen in the field "Any other information on materials and methods incl. tables" below (Table 1).

ACCEPTABILITY OF THE ASSAY
The results are considered as valid if the following acceptance criteria are met:
The test item induces a physical change (colour or precipitation) in the CDS solution in the qualify test.
The negative control does not induce a physical change (colour or precipitation) < 60 minutes in the CDS solution in the classification test.
The positive control induces a physical change (colour or precipitation) in the CDS solution in the classification test after 0 – 3 minutes.
Irritation / corrosion parameter:
other: other: time interval of colour change of CDS reagent after treatment of bio-barrier
Value:
0
Remarks on result:
other:
Remarks:
Basis: other: minutes (mean of 4 replicates). Time point: after 60 minutes of treatment. Remarks: A change of colour of CDS reagent after treatment of bio-barriers with test item was not observed during the test, i.e. for 60 minutes. . (migrated information)
Irritant / corrosive response data:
A change of colour of the CDS reagent after treatment of the bio-barriers with the test item was not observed up to 60 minutes. According to the classification criteria the test item was classified as non corrosive.

HISTORICAL DATA:

Table: Historical data

Mean time till colour change of positive control [min]

1.313

Standard Deviation [min]

0.45

Data of 46 studies performed from 2006 till 2012.

QUALIFY TEST:

The test item induced a change in colour in the qualify test after 1 minute incubation. Since a change in colour was visible in the “Qualify Test Vial”, the test item was considered to be suitable for the next step.

CATEGORISATION TEST:

Since the pH value of the test item in the “Category A Vial” was 7.5 and therefore above 7 the test item was assigned to category 2.

CLASSIFICATION TEST:

 

Test Group

 

 

Time Interval of
Colour Change

 

 

UN Packaging Group

 

 

R-Sentence

 

 

GHSand Regulation (EC) No 1272/2008 (CLP)

 

 

Negative Control

 

 

Colour change was not observed for 60 minutes

 

-

-

-

 

Positive Control

 

 

1 minute

 

 

I

 

 

R35

 

 

1A

 

 

Test Item

 

 

Colour change was not observed for 60 minutes

 

 

Non-corrosive

 

 

 

The test item was classified as non corrosive.

- The negative control did not induce a change in the colour of the CDS reagent after 60 minutes. The positive control showed a distinct change in the colour of the CDS reagent in the time interval of 0 – 3 minutes (after 1 minute). These results ensured the validity of the test.

Interpretation of results:
other: the test item is not corrosive.
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 2-ethylhexanoic acid, molybdenum salt is not corrosive to skin.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not classified as corrosive to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not classified as corrosive to the skin.
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 7 - 8 months
- Weight at study initiation: 2.8 - 3.2 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:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A dose of 0.5 mL of the test item was applied to the test site.
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:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Remarks:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Irritant / corrosive response data:
None of the three rabbits exposed for 4 hours to 0.5 mL 2-ethylhexanoic acid, molybdenum 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-Commission directive 67/548/EEC and its subsequent amendments, 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

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-14 to 2013-02-18
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 rabbits
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.66 or 2.68 kg
- Housing: the animals were individually housed in suspended cages.
- 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 animals were 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 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: 7 days
Remarks on result:
other: Initial pain reaction: practically no initial pain was observed. Area of cornea involved (opacity): one quarter (or less) but not zero (observed from 1 hour to 72 hours).
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 7 days
Remarks on result:
other: Slight inflammation of iris was observed at the 1 hour observation.
Remarks on result:
other: Severe to slight discharge was observed from the 1 hour observation to the 48 hours observation. Reddish brown coloured staining of the fur around treated eye was observed at the 1, 24, 48, and 72 hours as well as on day 7 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 observation. Slight conjunctivae redness was observed on Day 7.
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: Moderate chemosis was observed at the 1 hour observation. Slight chemosis was observed on Day 7.
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 cornea involved (opacity): one quarter (or less) but not zero (observed at the 1, 24 & 72 hours observation) & greater than one 1/4 but less than half (observed at the 48 hours observation).
Irritation parameter:
iris score
Basis:
mean
Remarks:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Remarks on result:
other: Slight inflammation of iris was observed at the 1 hour observation
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal #2
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 observation. Slight conjunctivae redness was observed on Day 7.
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 observation. Slight chemosis was observed on Day 7.
Irritant / corrosive response data:
Reddish brown coloured staining of the fur caused by the brown test item was noted around both treated eyes during the study.
Diffuse corneal opacity was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations.
Iridial inflammation was noted in both treated eyes one hour after treatment and in one treated eye at the 24 and 48-Hour observations. Iridial inflammation developed again in one treated eye at the 72-Hour observation.
Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 72-Hour observation with minimal conjunctival irritation noted in both treated eyes 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 body weight during the study.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
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.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
The test item was tested undiluted.
Duration of treatment / exposure:
10 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 L-glutamine, Na-bicarbonate and Taurine. 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 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. 141112)) or positive control (2-Ethoxyethanol (Sigma, 82024 Taufkirchen, Germany, lot no. BCBD1053V)) at a volume of 0.75 mL on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath, while the corneae were in a horizontal position. The test item, positive control and negative control were tested in triplicate.
The incubation time lasted ten 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. The corneae were then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a 2nd opacity reading (t130).
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. 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 followed by two hours incubation (t130).

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 (t130 – 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 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:
22.16
Remarks on result:
other: 10 min
Other effects / acceptance of results:
Relative to the negative control, the test item 2-ethylhexanoic acid, molybdenum salt induced an increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 22.16 (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 10 minutes incubation time

Test group

Opacity value = Difference (t130 – 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

0.050

 

0.050

1.75

 

1.75

Non corrosive / non severe irritant

0

0.051

0.76

2

0.048

2.73

Positive control

53.00*

0.818*

65.27

 

78.42

Corrosive / severe irritant

61.00*

0.914*

74.70

79.00*

1.085*

95.28

2-ethylhexanoic acid, molybdenum salt

21.00*

0.190*

23.86

22.16

Non corrosive / non severe irritant

18.00*

0.208*

21.13

19.00*

0.166*

21.50

* correctde values

- With the negative control neither an increase of opacity nor permeability of the corneae could be observed (meanin vitroirritation score 1.75).

- The positive control (2-Ethoxyethanol) caused clear opacity and distinctive permeability to the corneae (meanin vitroirritation score 78.42) 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

72.88

1.08

Standard Deviation

20.11

0.81

 Range of in vitro irritation scores 46.48 - 116.40  0.00 - 2.80

Values of 168 studies with liquid 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, molybdenum salt is not corrosive / not severe irritant to the eye (CLP/EPA/GHS (Cat 1)).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation in vivo


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.


 


Furthermore, a reliable in vitro study described by Heppenheimer (2013)(OECD 435; GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be corrosive 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) (section 5C4.1) (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 (2013)(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: 0.33 and 0.67

conjunctival redness: 2 for both animals

chemosis: 2 and 1.67

Reddish brown coloured staining of the fur around treated eye was observed in both animals at the 1, 24, 48, and 72 hours as well as on day 7 observation. 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.