Registration Dossier

Administrative data

Description of key information

Skin corrosion (OECD 431), EpiDermTM: non-corrosive
Skin irritation (OECD 439), EpiSkin: not irritating
Eye irritation (OECD 437), BCOP: corrosive

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
13 Mar 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted in 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Attenberger Fleisch GmbH&Co. KG, München, Germany
- Donor animals: no details given
- Date and time of eye collection: directly before the start of the experiment
- Time interval prior to initiating testing: no details given
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) on ice with penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: MC2 (Clermont, France)
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: RPMI with and without phenol red, supplemented with 1% [v/v] fetal calf serum and 2 mM L-glutamine; prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: initial opacity of 2 (corneas with an initial opacity above 7 in the opacitometer or with any macroscopic defects were not used)

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer and was read against an air-filled chamber.
- Specification of the device: MC2, Clermont, France

Vehicle:
physiological saline
Controls:
other: number of eyes for the negative control: 3; number of eyes for the positive control: 3
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20%

VEHICLE
- Substance: physiol. saline
- Concentration: 0.9% NaCl solution in deionised water
- Amount applied: 750 µL

POSITIVE SUBSTANCE
- Substance: imidazole in physiol. saline
- Concentration: 20% imidazole in 0.9% NaCl solution in deionised water
- Amount applied: 750 µL
Duration of treatment / exposure:
4 h at 32 ± 1 °C
Number of animals or in vitro replicates:
Number of eyes for the test item: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed at least three times.
- Medium for washing the corneas: MEM containing phenol red
- Medium for final rinsing: RPMI without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Time of determination: Directly after refilling complete RPMI without phenol red in the anterior chamber the final opacity was measured.
- Specification of the device: MC2, Clermont, France

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: 90 min at 32 ± 1 °C
- Treatment for measuring: OD490 of a 1 mL aliquot was determined.
- Dilution of the medium: undiluted
- Specification of the spectrophotometer: Jenway, UK
Irritation parameter:
in vitro irritation score
Remarks:
mean out of all 3 eyes
Run / experiment:
4 h exposure
Value:
235.99
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
An IVIS of 235.99 was observed for the test substance indicating that the test susbstance has corrosive or severe irritant properties.

Table 1: Opacity values

Parameter

Initial opacity

Final opacity

Opacity change

Mean opacity change of NC

Corrected opacity change

Mean opacity value

Negative control

2

1

-1

-0.67

-

-

2

1

-1

 

2

2

0

 

Test substance

1

216

215

-

215.67

236

2

266

264

264.67

2

229

227

227.67

Positive control

3

162

159

-

159.67

158

3

194

191

191.67

3

125

122

122.67

 

 

Table 2: Permeability values (optical density (OD) at 490 nm)

Parameter

OD490

Mean OD490 value

Corrected OD490 values

Mean Corrected OD490 values

Negative control

0.010

0.010

-

-

0.007

0.012

Test substance

0.003

0.009

-0.007

-0.001

0.012

0.002

0.012

0.002

Positive control

1.702

1.651

1.692

1.641

1.511

1.501

1.740

1.730

 

 

Table 3: In-Vitro Irritancy Score (IVIS) values

 

Mean IVIS

 

Test substance

235.99

Positive control

182.62

 

 

 

Interpretation of results:
other: CLP/EU GHS Category 1 according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: Cat. 1, H318
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid was tested for its skin corrosion potential in the EpiDermTMSkin Model, which is a three-dimensional in-vitro model of the human epidermis and is used to distinguish between corrosive and non-corrosive chemicals (Lehmeier, 2012a). The skin corrosion test was performed according to OECD guideline 431 and in compliance with GLP. 25 mg Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (moistened with water) was applied to the intact reconstructed human epidermis for 3 and 60 min, respectively. Duplicate cultures were used for each group and treatment period. Concurrent control tissues treated with A. dest. served as negative controls, positive controls were exposed to 8N KOH. After the treatment the test item was washed from the skin surface with phosphate-buffered saline. For determining alterations in cell viability, MTT reduction assays were performed. The relative cell viability for the test item was 93% and 88% after 3 and 60 min, respectively. Therefore, the test substance was considered to be non-corrosive as the evaluation criteria for non-corrosivity (≥50% viability after 3 min and ≥15% viability after 60 min) were met. After treatment with the positive control substance KOH the viability was reduced to 14% and 6% after 3 and 60 min, respectively. The positive control met the criteria for corrosivity (<50% viability after 3 min). In conclusion, the test substance was not corrosive under the conditions of the study.

In concordance with the Integrated Testing Strategy (ITS) described in the ECHA guidance document (ECHA, 2013) an in-vitro skin irritation test was performed with Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid according to OECD guideline 439 and in compliance with GLP in a reconstructed three-dimensional human epidermis model (EpiskinSM) (Lehmeier, 2012b). 10 mg Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (moistened with water) was applied to the intact reconstructed human epidermis for 15 min. After treatment the skin was washed with phosphate-buffered saline (PBS) and was further incubated for 42 h. Triplicate cultures were used for each test or control group. Concurrent control tissues treated with PBS served as negative controls, positive controls were exposed to 5% SDS. For determining alterations in cell viability, MTT reduction assays were performed.

The relative cell viability was 112% and 5% for the test item and the positive control, respectively. The positive control met the criteria as an irritant (≤50% viability after 15 min treatment). The test item met the criteria as not irritant (>50% viability after 15 min treatment). The test acceptance criteria were met, therefore the test substance is considered to be not irritant under the conditions of the study.

In conclusion, according to the ITS for irritation/corrosion (ECHA, 2013) Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid is considered to be not irritant to skin as validated in-vitro skin corrosion and skin irritation tests were negative and no other information is available indicating that the substance is irritating to skin.

Eye irritation

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid was tested for its eye irritation potential in a study according to OECD guideline 437 (BCOP) in bovine eyes (Lütkenhaus, 2012). Fresh eyes were collected at the day of the experiment and were prepared before use. Corneas were dissected with a 2-3 mm rim of sclera and were mounted in cornea holders (3 corneas per group). 750 µl of a test substance solution (20% in physiological saline) was applied to the cornea and incubated for 4 h at 32°C (closed-chamber method). 750 µL of a 20% imidazole solution was used as a positive control. After treatment the corneas were washed with MEM containing phenol red and the corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer. For assessment of the corneal permeability sodium fluorescein (5 mg/mL) solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber (within 90 min at 32°C) was quantitatively measured via UV/VIS spectrophotometry at 490 nm recorded as optical density (OD490). The mean opacity value, mean corrected OD490 value and the in-vitro irritancy score (IVIS) for the test substance were 236, -0.001, and 235.99, respectively. For the positive control the mean opacity value, mean corrected OD490 value and the in-vitro irritancy score were 158, 1.641, and 182.62. IVIS ≥55 indicate corrosive/severe irritant properties according to CLP regulation. In conclusion, the test substance was corrosive/severely irritant under the conditions of the study, and has to be classified as Cat 1 (H318) according to CLP regulation.

Justification for classification or non-classification

The available data on skin irritation of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.

The available data on eye irritation of the Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid meet the criteria for classification as 'Causes serious eye damage: Cat 1 (H318) according to Regulation (EC) 1272/2008.