Reaction mass of octan-2-ylbenzene, octan-3-ylbenzene and octan-4-ylbenzene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April - 05 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(octan-2-yl)benzene

EC Number:

950-576-0

Cas Number:

31047-57-1

Molecular formula:

C14H22

IUPAC Name:

(octan-2-yl)benzene

Test material form:

liquid

Details on test material:

Identification: SHR 1396
Physical Description: Clear colorless liquid
Storage Conditions: At room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): Lot no.: 30903 kit A and kit B
- Production date: 3 July 2019
- Date of initiation of testing: 30 April

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 62 - 95%, containing 5.0 ± 0.5% CO2 in air in the dark at 36.3 - 37.1°C.
- Temperature of post-treatment incubation: 37.0 ± 1.0ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one step, washing done with PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
- 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to SHR 1396 and two for a 1-hour exposure.
- For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes treatment

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability: 7.4%

Remarks on result:

other: not corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability: 5.7%

Remarks on result:

other: not corrosive

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 5.7%.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean Absorption values

 

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.647	1.657	1.652	±	0.007	1.788	1.559	1.673	±	0.162
SHR 1396	1.800	1.727	1.763	±	0.051	1.715	1.677	1.696	±	0.027
Positive control	0.113	0.131	0.122	±	0.013	0.086	0.103	0.095	±	0.012

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.045). Isopropanol was used to measure the background absorption.

       
Table 2: Mean Tissue Viability

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
SHR 1396	107	101
Positive control	7.4	5.7

        
Table 3: Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative control	0.6	13
SHR 1396	4.0	2.2
Positive control	14	16

CV (%) = 100 - [(lowest OD₅₇₀/highest OD₅₇₀) x 100%]

Table 4: Individual OD measurements

	3-minute application (OD570)        A               B		1-hour application (OD570)        A               B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.6880	1.6929	1.8350	1.6080
	1.6925	1.7053	1.8375	1.5989
	1.6959	1.7082	1.8254	1.6037
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3
	1.8530	1.7640	1.7682	1.7250
	1.8358	1.7813	1.7528	1.7271
	1.8444	1.7710	1.7578	1.7123
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3
	0.1580	0.1774	0.1313	0.1477
	0.1581	0.1761	0.1316	0.1482
	0.1580	0.1754	0.1308	0.1469

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 5: Historical control data

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258 – 2.615	1.371 – 2.371	0.092 – 0.56	0.046 – 0.339
Mean	1.73	1.78	0.19	0.14
SD	0.24	0.21	0.09	0.05
n	116	119	114	112

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2015 to November 2018.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The results of an in vitro skin corrosion test showed that SHR 1396 was not corrosive to the skin.

Executive summary:

An in vitro skin corrosion test was performed following OECD guidelines and GLP principles. The test item SHR 1396 was applied undiluted (50 µL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5.7% after the 1-hour exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%,indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with SHR 1396 compared to the negative control tissues was 107% and 101%, respectively. Because the mean relative tissue viability for SHR 1396 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment SHR 1396 is considered to be not corrosive and does not need to be classified.