4-methyl-4-phenylpentan-2-one

Endpoint summary

Description of key information

Skin irritation/corrosion (OECD 439 and OECD 431): irritating

Eye irritation (OECD 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb - 02 Jun 2017

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 28 Jul 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation No 440/2008 of 30 May 2008, 1st ATP of 23 Jul 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23395
- Delivery date: 31 May 2016
- Date of initiation of testing: 21 Feb 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.211 ± 0.175 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 4.83 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less or equal than 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

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

approximately 42 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

15.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance was not considered to be a MTT reducer.
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.978, 1.710 and 1.638) was in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance to 3.1% as compared to the negative control thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance and negative control in the main test were below 10.4% (threshold of OECD 439: <18%).

Table 2. Results after treatment with the test substance and controls

Test group	Mean absorbance at 570 nm*			Rel. absorbance (%)**			Rel. SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3	Tissue 1	Tissue 2	Tissue 3
Negative control	1.978	1.710	1.638	111.4	96.3	92.3	10.1	100.0
Positive control	0.057	0.056	0.053	3.2	3.1	3.0	4.1	3.1
Test substance	0.312	0.264	0.260	17.6	14.9	14.7	10.4	15.7

* Mean of three replicate wells after blank correction (blank = 0.036)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Interpretation of results:

other: irritating potential (Skin Irrit. 2 or Skin Corr. 1 according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the conducted test, the test substance possessed irritating properties towards reconstructed human epidermis tissue in the EpiDerm™ model.

Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2017). After treatment with the test substance for 60 min the tissue viability decreased to 15.7% compared to the negative control (threshold for irritancy≤50%).Therefore, the test substance is considered to possess an irritant potential towards human-derived epidermal keratinocytes.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 21 Apr 2017

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 29 Jul 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU B.40 bis (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE)
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 25806
- Delivery date: 19 Apr 2017
- Date of initiation of testing: 19 Apr 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure), 37 ± 1.5 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS (20 times) in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v.4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm™ tissue was assessed by an MTT cell viability test. The determined OD (540 - 570 nm) was 1.581 ± 0.238 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.58 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm™ tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce the MTT solution, an additional functional check was not performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: single experiment

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 ± 0.5 min and 60 ± 5 min

Number of replicates:

duplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

109.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

114.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: Since the test substance did not directly reduce MTT, an additional test with freeze-killed or viable tissues was not performed.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and thus passed the colour interference pre-test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.559 and 1.661).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control for 1 hour was <15% compared to the negative control (9.6%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 0.6% and 9.7%).

Table 2. Results after treatment with the test substance and controls

	Exposure interval (min)	Aborbance at 570 nm *		Mean absorbance of 2 tissues	Absorbance (% of negative control) **		CV (%)	Rel. absorbance (% of negative control) **
		Tissue 1	Tissue 2		Tissue 1	Tissue 2
Negative control	3	1.522	1.586	1.554	97.9	102.1	2.9	100.0
Positive control		0.348	0.399	0.373	22.4	25.7	9.7	24.0
Test substance		1.681	1.731	1.706	108.1	111.4	2.1	109.8
Negative control	60	1.624	1.555	1.590	102.2	97.8	3.1	100.0
Positive control		0.154	0.151	0.153	9.7	9.5	1.2	9.6
Test substance		1.807	1.822	1.814	113.7	114.6	0.6	114.1

* Mean of three replicate wells after blank correction (blank: 0.037)

 ** Relative absorbance (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Interpretation of results:

other: non-corrosive according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the EpiDerm™ model.

Executive summary:

The skin corrosion potential of the test substance was investigated by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2017). After treatment with the test substance the tissue viability did not decrease (3 min exposure: 109.8%; 1 h exposure:114.1%) compared to the negative control. Therefore, the test substance is not considered to be corrosive towards human-derived epidermal keratinocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

20 Feb 2017

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

(July 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in Hank's Buffered Salt Solution (HBSS) containing 1% (v/v) Penicillin/Streptomycin (100 units/mL, respectively).
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and directly used in the BCOP test.
- 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL

NEGATIVE CONTROL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 cornea

Run / experiment:

10 min exposure

Value:

0.18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed.
The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 79.74) corresponding to a classification as serious eye damaging.
Relative to the negative control, the test substance did not cause an increase in corneal opacity or permeability (mean IVIS = 0.18).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for postive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

 Table 2. Results after 10 min incubation time.

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative Control	0	0.00	0.064	0.061	0.96	0.92
	0		0.061		0.92
	0		0.058		0.87
Positive Control	61.0*		1.072*		77.08	79.74
	60.0*		0.957*		74.36
	66.0*		1.452*		87.78
Test substance	0.00*		0.007*		0.11	0.18
	0.00*		0.021*		0.32
	0.00*		0.008*		0.12

* Corrected values

Interpretation of results:

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

Conclusions:

Under conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test subtance was not irritating to the eye.

Executive summary:

The eye irritation potential of the test substance was determined by a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2017). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.18. Thus, the test substance is not considered to be irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The skin irritation potential of the test substance was determined by an in vitro skin irritation test using a reconstructed human skin model according to OECD Guideline 439 and in compliance with GLP (2017). After treatment with the test substance for 60 min the tissue viability decreased to 15.7% compared to the negative control (threshold for irritancy ≤ 50%).Therefore, the test substance is considered to possess an irritant potential towards human-derived epidermal keratinocytes.

In a next step, the skin corrosion potential of the test substance was investigated by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2017). After treatment with the test substance the tissue viability did not decrease (3 min exposure: 109.8%; 1 h exposure: 114.1%) compared to the negative control. Therefore, the test substance is not considered to be corrosive towards human-derived epidermal keratinocytes.

In conclusion, based on the available data on skin irritation/corrosion, the test substance is considered to be irritating to skin and therefore classified as Skin Irrit. 2 (H315) according to CLP.

Eye

The eye irritation potential of the test substance was determined by a bovine corneal opacity and permeability (BCOP) test according to OECD Guideline 437 and in compliance with GLP (2017). Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.18. Thus, the test substance is not considered to be irritant to the eye.

Justification for classification or non-classification

The available data on skin irritation meet the criteria for classification as Skin Irrit. 2 (H315) according to Regulation (EC) 1272/2008.

The available data on eye irritation do not met criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.