γ-valerolactone

Endpoint summary

Description of key information

Skin irritation/corrosion

Two in vitro assays were part of an in vitro skin irritation and corrosion test strategy. A Skin Corrosion Test according to OECD guideline 431 showed no skin damaging properties of the substance, whereas a Skin Irritation Test according to OECD guideline 439 showed a skin irritating potential. It was concluded that the test item is a skin irritant.

 

Eye irritation/corrosion

Two in vitro assays were part of an in vitro eye irritation and corrosion test strategy. A Eye Corrosion Test according to OECD guideline 437 showed no eye damaging properties of the substance, whereas an Eye Irritation Test according to OECD guideline 492 showed an eye irritating potential. It was concluded that the test item is an eye irritant.

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:

2020-07-13 to 2020-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

31 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not indicated

Justification for test system used:

OECD accepted in vitro model system as part of a turnkey test strategy to assess the skin irritation potential of chemicals

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)
- Tissue batch number: 30881
- Date of initiation of testing: 2020-07-22 (Date of quality check)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (for the 3-minutes incubation period) or at 37°C (for 1-hour incubation period)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were washed with sterile PBS (number and volume not indicated)
- Observable damage in the tissue due to washing: Not indicated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL of a 1.0 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability and barrier function: MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within an established range based on a historical database of results (table 1)

NUMBER OF REPLICATE TISSUES: 2 tissues per incubation time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MTT interference occured, thus no freeze-killed control tissues were used.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One test sequence was conduced using 2 replicate tissues per time point

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 50 µL deionized water

POSITIVE CONTROL
- Amount applied: 50 µL 8 N potassium hydroxide solution
- Concentration: 8 N

Duration of treatment / exposure:

3 min and 1 hour treatments

Duration of post-treatment incubation (if applicable):

No post-incubation period

Number of replicates:

2 tissue replicates per time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 (1 hour incubation time, 2 tissue replicates)

Value:

21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 (3 minutes incubation time, 2 tissue replicates)

Value:

86.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (see table 2-4)
- Acceptance criteria met for positive control: Yes (see table 2-4)
- Acceptance criteria met for variability between replicate measurements: Yes (see table 2-4)
- Range of historical values if different from the ones specified in the test guideline: No

Table 3: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficients of variation (exposure period 3 min)

Test substance identification		tissue 1	tissue 2	mean	SD	CV [%]
NC	mean OD570	2.085	2.131	2.108
	Viability [% of NC]	98.9	101.1	100.0	1.5	1.5
Test item	mean OD570	1.735	1.916	1.825
	Viability [% of NC]	82.3	90.9	86.6	6.1	7.0
PC	mean OD570	0.171	0.192	0.181
	Viability [% of NC]	8.1	9.1	8.6	0.7	8.0

 Table 4: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficients of variation (exposure period 1 hour)

Test substance identification		tissue 1	tissue 2	mean	SD	CV [%]
NC	mean OD570	2.083	1.936	2.010
	Viability [% of NC]	103.6	96.4	100.0	5.2	5.2
Test item	mean OD570	0.440	0.403	0.421
	Viability [% of NC]	21.9	20.0	21.0	1.3	6.2
PC	mean OD570	0.061	0.063	0.062
	Viability [% of NC]	3.0	3.1	3.1	0.1	2.3

Interpretation of results:

GHS criteria not met

Remarks:

The Skin Corrosion Test according to OECD guideline 431 showed no skin corrosive potential of gamma-Valerolacton.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-07-13 to 2020-12-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

31 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not indicated

Justification for test system used:

OECD accepted in vitro model system as part of a turnkey test strategy to assess the skin irritation potential of chemicals

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia)
- Tissue batch number: 30881
- Date of initiation of testing: 2020-07-22 (Date of quality check)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (for 25 min), thereafter at 37°C (for 35 min)
- Temperature of post-treatment incubation: 37°C (for 24 ± 2 hours and additional 18 ± 2 hours)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were washed with sterile PBS (number and volume not indicated)
- Observable damage in the tissue due to washing: Not indicated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mL of a 1.0 mg/mL MTT solution
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability and barrier function: MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within an established range based on a historical database of results (table 1)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MTT interference occured, thus no freeze-killed control tissues were used.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One test sequence was conduced using 3 replicate tissues

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-irritating to skin if the mean tissue viability after 1 hour exposure is greater than 50%.
- The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and posttreatment incubation is less than or equal (≤) to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL PBS

POSITIVE CONTROL
- Amount applied: 30 µL 5% SDS
- Concentration: 5%

Duration of treatment / exposure:

1 hour treatment, followed by a 42-hour post-incubation period

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 tissue replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1 (mean of 3 tissues)

Value:

4.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Not indicated
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (see table 2+3)
- Acceptance criteria met for positive control: Yes (see table 2+3)
- Acceptance criteria met for variability between replicate measurements: Yes (see table 2+3)
- Range of historical values if different from the ones specified in the test guideline: No

Table 3: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficients of variation

Test substance identification		tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC	mean OD570	1.999	1.887	1.933	1.940
	Viability [% of NC]	103.1	97.3	99.7	100.0	2.9	2.9
Test item	mean OD570	0.075	0.091	0.117	0.094
	Viability [% of NC]	3.9	4.7	6.0	4.9	1.1	22.7
PC	mean OD570	0.041	0.037	0.036	0.038
	Viability [% of NC]	2.1	1.9	1.9	2.0	0.1	6.9

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

The Skin Irritation Test according to OECD guideline 439 showed a skin irritating potential of gamma-Valerolacton

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-07-13 to 2020-11-17

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: B.69 Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage; Official Journal of the European Union, No.L 247

Version / remarks:

31 July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: OECD accepted in vitro model as part of a turnkey test strategy to assess the eye irritation potential of chemicals
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) and are available commercially as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose. The cells used to produce EpiOcular tissue are screened for potential biological contaminants. No contaminations have been detected in this tissue batch.
- RhCE tissue used, including batch number: All cells used to produce EpiOcular are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions (keratinocyte strain 4F1188, Lot No 30669)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

After 12 minutes of post-soak immersion, the tissues were incubated at standard culture conditions for 2 hours (post-incubation period)

Number of animals or in vitro replicates:

a single test composed of 2 tissue replicates

Details on study design:

- Details of the test procedure used: Using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were applied concurrently with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
- Doses of test chemical and control substances used: 50 µL of test chemical/positive control/negative control
- Duration and temperature of exposure and post-exposure incubation periods: Exposure at 37°C for 30 minutes, then post-exposure incubation for 2 hours at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Prior to testing, the test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
- Number of tissue replicates used per test chemical and controls: 2 replicates per test item/positive control/negative control. Freeze-killed control tissues were not tested as no direct reduction of MTT by the test substance occured.
- Wavelength used for quantifying MTT formazan: Measurement using a filter wavelength 570 nm without reference filter using a SunriseTM Absorbance Reader
- Description of the method used to quantify MTT formazan: The OD570 values determined for the various tissues are a measure of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Please refer to table 1
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Please refer to table 2+3
- Reference to historical data of the RhCE tissue construct: Please refer to table 3
- Positive and negative control means and acceptance ranges based on historical data: Please refer to table 2+3
- Acceptable variability between tissue replicates for positive and negative controls: Please refer to table 2
- Acceptable variability between tissue replicates for the test chemical: Please refer to table 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Run 1 (mean of 2 replicate tissues)

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: No

 

Table 4: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	2.300	2.383	2.342
	vability [% of NC]	98.2	101.8	100.0	3.5
Test item	mean OD570	0.071	0.045	0.058
	vability [% of NC]	3.0	1.9	2.5	1.1
PC	mean OD570	0.502	0.382	0.442
	vability [% of NC]	21.4	16.3	18.9	5.1

 

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

The RhCE Test according to OECD guideline 492 showed an eye irritating potential of gamma-Valerolacton.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

09 December 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: 1% (v/v) penicillin/streptomycin in the HBSS storage solution
- Quality check of the isolated corneas: Only corneae with a value of the basal opacity < 7 were used.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

Ten minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

Two hours

Number of animals or in vitro replicates:

3 corneas/negative control, 3 corneas/positive control and 3 corneas/test item

Details on study design:

NUMBER OF REPLICATES
3 corneaes per test item treatment/ positive control/ negative control

NEGATIVE CONTROL USED
Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED
2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL, ten minutes (± 30 seconds) exposure

POST-INCUBATION PERIOD: Two hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microtiter plate reader at OD490

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIScorr = (opacity value – mean of opacity of negative control) + 15 x (permeability value – mean permeability of the negative control)

DECISION CRITERIA
Decision criteria as indicated in the test guideline were used (see table 1)

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Run 1 (mean of three corneas)

Value:

40.46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, see "Any other information on materials and methods incl. tables"
- Acceptance criteria met for positive control: Yes, see "Any other information on materials and methods incl. tables"
- Range of historical values if different from the ones specified in the test guideline: No

Table 3: Results after 10 min treatment time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS
		Mean		Mean
	0		0.055		0.83
Negative Control	0	0.00	0.048	0.052	0.72	0.79	0.06
	0		0.054		0.81
	83.00*		0.587*		91.80
Positive Control	90.00*		0.599*		98.98	93.56	4.79
	83.00*		0.460*		89.90
	34.00*		0.652*		43.78
Test item	27.00*		0.569*		35.53	40.46	4.36
	33.00*		0.606*		42.09

*corrected values

Interpretation of results:

GHS criteria not met

Remarks:

The BCOP eye corrosion Test according to OECD guideline 437 showed no eye corrosive potential of gamma-Valerolacton

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro, RL1

The objective was to assess the skin irritation potential of the test item. The potential of the test substance to cause dermal irritation was assessed in a GLP compliant OECD 439 study, by a single topical application of 30 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The skin irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. In addition to the test substance, 30 μL per tissue of a negative control (PBS) and of a positive control (5% SDS) were applied to three tissues each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced. The relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 4.9%. The variability between the results of the tissues is within the acceptance range. The tissues treated with the positive control (5% SDS) showed a relative mean viability of 2.0% reflecting the expected sensitivity of the tissues. The mean OD570 of the negative control (PBS) fulfills the acceptance criteria and demonstrates the validity of the assay. According to OECD 439, the test item shows a skin irritation potential (BASF, 2020).

 

Skin irritation in vitro, RL1

The objective was to assess the skin corrosion potential of the test item. The potential of the test substance to cause dermal corrosion was assessed in a GLP compliant OECD 431 study, by a single topical application of 50 μL undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. According to OECD 431, the test item does not show skin corrosion (BASF 2020).

 

Conclusion: Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy. Based on the results observed and by applying the evaluation criteria, it was concluded that the test item shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

 

Skin irritation, human, RL4 (please refer to section 7.10.5)

In a study in humans, 10% of the test item in diethyl phthalate produced no irritation after 48 hours under occlusion (Opdyke 1962-1980, original study by Kligman, 1978).

 

Skin irritation, rabbit, RL4

The test item applied undiluted to intact or abraded rabbit skin for 24 hours under occlusion was slightly irritating (Opdyke 1962-1980, original study by Moreno, 1978).

 

Eye irritation in vitro, EIT, RL1

The potential of the test item to cause ocular irritation was assessed in a GLP compliant OECD 492 study, by a single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each. Tissue destruction was determined by measuring the metabolic activity of the tissues after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was not introduced. The relative mean viability of the tissues treated with the test substance was 2.5%. The variability between the results of the tissues is within the acceptance range. The tissues treated with the positive control (methyl acetate) showed a relative mean viability of 18.9% reflecting the expected sensitivity of the tissues. The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria and demonstrates the validity of the assay. Based on the results observed and by applying the evaluation criteria, it was concluded that gamma-Valerolactone shows an eye irritation potential in the EpiOcular™ in vitro eye irritation test under the test conditions chosen. The test method does not allow for the evaluation of serious eye damage. The result does not exclude a serious eye irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed (BASF, 2020).

 

Eye corrosion ex vivo, BCOP, RL1

The GLP compliant in vitro study according to OECD guideline 492 was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item gamma-Valerolactone caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 40.46. According to OECD 437 the test item does not show serious eye damage. The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed (BASF, 2021).

 

Conclusion: Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of eye irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro eye irritation and corrosion test strategy. Based on the results of the BCOP and EpiOcular tests, it was concluded that the test item shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

Using a weight of evidence approach, the skin and eye irritation potential of the test item was assessed. The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item was found to be irritating to skin and eye, thus, the test item is considered to be classified for skin and eye irritation under Regulation (EC) No 1272/2008, as amended for fifteenth time in Regulation (EU) No 2020/217.