Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

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-March-2021 to 02-April-2021 (experimental dates)

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30-May-2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: beige powder

Specific details on test material used for the study:

Appearance: Beige Powder
Storage conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm™ Human Skin Model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Skin from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ Reconstructed Human Epidermis was supplied by MatTek In Vitro Life Sciences Laboratories, Lot number 34139 (keratinocyte strain: 00267). In order to test if the test item has potential to interfere with the MTT endpoint, a pre-test was carried out to check for the ability of the test item to directly reduce MTT and if it would become colored in wet or aqueous conditions.

Main test
Pre-incubation: 0.9 mL of the assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37°C, 5% CO2) for approximately 1 hour before dosing.

Application of test Item and rinsing:
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 25 mg of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 µL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37°C, 5% CO2) for the 60 Minute exposure period. When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure.
Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds. They were then blotted and transferred to the 24 well plate prepared for MTT-loading. The plate was incubated (37°C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.

After the 3 Hour MTT incubation was complete, the tissue inserts were blotted and transferred to 24 well plates for formazan (reduced MTT) extraction. The formazan was extracted from the top and bottom of the tissue by completely immersing the tissue insert in 2 mL of isopropanol. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96 well plate. The optical density (OD) was measured at 570 nm (OD570).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

• Test item: 25 mg BTMR
• Positive control: 50 µL of 8.0 N Potassium Hydroxide
• Negative control: 50 µL of sterile distilled water

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

99.7

Negative controls validity:

valid

Remarks:

100*

Positive controls validity:

valid

Remarks:

3.1

Remarks on result:

other: *The mean viability of the negative control tissues is set at 100%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

94.3

Negative controls validity:

valid

Remarks:

100*

Positive controls validity:

valid

Remarks:

3.3

Remarks on result:

other: *The mean viability of the negative control tissues is set at 100%

Other effects / acceptance of results:

The MTT solution containing the test item did not turn blue/purple. This was taken to indicate the test item did not reduce MTT. The solution containing the test item did not become colored and presented a separate layer of insoluble beige powder. This color was attributed to the intrinsic color of the test item itself and not present in the solution. It was therefore unnecessary to run color correction tissues.

The mean OD570 for the negative control treated tissues was 2.049 for the 3 Minute exposure period and 2.115 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1      Mean OD₅₇₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Exposure Period (minutes)	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control
3	1.996	2.049	0.075	3.7	100*
3	2.102	2.049	0.075	3.7	100*
60	2.148	2.115	0.047	2.2	100*
60	2.081	2.115	0.047	2.2	100*
Positive Control
3	0.074	0.064	0.014	na	3.1
3	0.054	0.064	0.014	na	3.1
60	0.080	0.070	0.014	na	3.3
60	0.060	0.070	0.014	na	3.3
Test Item
3	2.090	2.042	0.068	3.3	99.7
3	1.994	2.042	0.068	3.3	99.7
60	2.045	1.995	0.071	3.6	94.3
60	1.944	1.995	0.071	3.6	94.3

OD =  Optical density

* =           The mean percentage viability of the negative control tissue is set at 100%

na =          Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of an in vitro corrosivity test, performed according to OECD/EC guidelines and GLP principles, BMTR was considered to be non-corrosive to the skin.

Executive summary:

The corrosivity potential of BTMR was assessed item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. Testing was done according to OECD/EC guidelines and GLP principles.

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. 

The mean viability percentage after 3 minute expsoure was 100, 3.1 and 99.7 for negative control, positive control and test item, respectively. For the exposure period of 60 minutes, the mean viability percentage was found to be 100, 3.3 and 94.3 for negative control, positive control and test item, respectively. 

The criteria required for acceptance of results in the test were satisfied.

In conclusion, BTMR was considered to be non-corrosive to the skin under the experimental conditions reported in this study.