Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

Endpoint summary

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

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm, reconstructed three-dimensional human epidermis (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
EpiDermTMSCIT (EPI-200):
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 3 and 60 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 550 nm wave length in a plate spectrophotometer.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg
- Concentration: 100% (moistened with 25 µL water)

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: Potassium hydroxide (8 N, CAS 1310-58-3, Lot 10357004)

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min of exposure

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min of exposure

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item the viability was 93% and 88% after treatment for 3 and 60 min, respectively. Therefore the test item is considered to be not corrosive.

Table 1: MTT assay after 3 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD550	2.088 2.085 2.082	1.947 1.985 1.978	0.309 0.315 0.312	0.269 0.271 0.269	2.001 1.907 1.878	1.803 1.859 1.818
OD550(mean)	2.085	1.970	0.312	0.270	1.929	1.827
SD	0.003	0.020	0.003	0.001	0.064	0.029
OD550(mean values of replicates)	2.028		0.291		1.878
Viability (%)	100		14		93
Mean inter tissue viability difference (%)	5.7		14.5		5.4

 

Table 2: MTT assays after 60 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD550	2.032 1.954 1.976	2.014 1.992 1.974	0.114 0.117 0.115	0.119 0.129 0.121	1.857 1.754 1.762	1.742 1.717 1.736
OD550(mean)	1.987	1.993	0.115	0.123	1.791	1.732
SD	0.040	0.020	0.002	0.005	0.057	0.013
OD550(mean values of replicates)	1.990		0.119		1.761
Viability (%)	100		6		88
Mean inter tissue viability difference (%)	0.3		6.4		3.4

 

Table 3: Historical data

	mean	SD	n
OD550of Negative Controls (3 min)	1.731	0.176	25
OD550of Negative Controls (60 min)	1.748	0.192	25
Relative tissue viability (%) of positive controls (3 min)	19.0	5.958	24
Max. inter tissue viability difference (%)	13.9	6.627	144

 

Interpretation of results:

other: non-corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted in 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiSkin, reconstructed three-dimensional human epidermis (EpiskinSM)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SKIN MODEL
- Source: SkinEthic laboratories, Lyon, France

TEST METHOD
EpiSkin small model (Batch No. 12-EKIN-013):
The model represents a reconstructed three-dimensional skin model based on normal adult human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a functional stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Upon receipt, tissues were transferred into 12-well plates containing 2 mL prewarmed assay medium per well and preincubated in a humidified incubator for at least 24 h (37 ± 1 °C, 5% CO2) before use.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum
TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 2 mL prewarmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After the 3 h MTT incubation period the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts were transferred into suitable tubes and 500 µL of acidic isopropanol were added. Extraction was carried out protected from light over the weekend at 2-8°C. At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous. If any visible cell/tissue fragments were in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings. Per each tissue 2x200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550m nmwithout reference wavelength in a plate spectrophotometer.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg
- Concentration: 100% (moistened with 10 µL water)

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: Sodium Dodecyl Sulfate (5%, SDS, CAS 151-21-3, Lot 9S009119)

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

The test was performed in triplicates for each test or control group.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min of exposure

Value:

112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item the viability was 112 after treatment for 15 min and a 42-h post-incubation period. Therefore the test item is considered to be not irritant.

Table 1: MTT assay after 15 min exposure

	Negative control			Positive control			Test item
Tissue sample	1	2	3	1	2	3	1	2	3
OD550	0.904 0.909	0.959 0.946	0.937 0.928	0.084 0.083	0.087 0.097	0.079 0.082	1.071 1.092	0.950 0.975	1.060 1.087
OD550(mean)	0.863	0.909	0.890	0.041	0.049	0.038	1.039	0.919	1.031
OD550(mean values of replicates)	0.887			0.042			0.996
SD	0.021			0.006			0.061
Viability (%)	100			5			112
Mean inter tissue viability difference (%)	2.6			14.0			6.7

 

Table 2: Quality criteria

	value	cut off	pass/fail
OD550blank	0.043	<0.1	pass
Absolute OD550of Negative Controls	0.930	0.6≤NC≤1.5	pass
Relative tissue viability (%) of positive controls	5.0	≤40	pass
Max. inter tissue viability difference (%)	0.7-7.5	<18	pass

 

Table 3: Historical data

	mean	SD	n
OD550of Negative Controls	0.44	0.002	20
Absolute OD550of Negative Controls	0.863	0.143	20
Relative tissue viability (%) of positive controls	10.2	6.030	20
Max. inter tissue viability difference (%)	5.3	3.742	62

 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

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

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. QA statement)

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 EpiDerm^TMSkin 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.