[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August - 29 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study conducted to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Molecular formula:

Not available: variable

IUPAC Name:

Reaction products of 2,3; 2,4; 2,5 and 2,6 mixed xylidenes, C7 and C8 linear substituted naphthols and diazonium salts

Test material form:

solid: particulate/powder

Details on test material:

Batch No. SR164:1
Appearance Dark red solid (see certificate of analysis)
Test substance storage Ambient conditions but protected from temperatures below 5°C

Stability under storage Stable under recommended storage conditions.
Manufacturing Date 12 July 2022
Expiry date 12 July 2027

Specific details on test material used for the study:

No further details in the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Human

Source strain:

other: Human

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 22 EKIN 034.
This model is a three-dimensional human epidermis model, which consists of adult humanderived
epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source
EPISKIN, Lyon, France.

Justification for test system used:

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 22 EKIN 034.
This model is a three-dimensional human epidermis model, which consists of adult humanderived
epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.


Source
EPISKIN, Lyon, France.
Experimental Design
Test for the Interference of the Test Material with the MTT Endpoint
A test material may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Material
The test material was checked for possible color interference before the study was started.
Some non-colored test materials may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of the test material or 50 μL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking.
At the end of the exposure time, the mixtures were centrifuged for 30 seconds at 16000 g of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
If after subtraction of the negative control, the OD for the test material solution is >0.08, the test material is considered as possibly interacting with the MTT measurement.

Test for Reduction of MTT by the Test Material
The test material was checked for possible direct MTT reduction before the study was started.
To assess the ability of the test material to reduce MTT, approximately 50 mg of the test material was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test material interacts with MTT.

Test System Set Up
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with
pre-warmed Maintenance Medium for 23 hours at 37°C. Maintenance medium and
Assay medium were supplied by EPISKIN, Lyon, France.
Killed tissues (EPISKIN-SMTM, 0.38 cm2, Lot no.: 22 EKIN 013, 22 EKIN 030.
Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored in a freezer set to maintain -15°C.
Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.
MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 1 mg/mL in PBS) diluted (3x) in Assay medium (final concentration 0.3 mg/mL).
Environmental conditions
All incubations, with the exception of the test material incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 44 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Material Preparation
The solid test material liquified by heating until 65.0 °C in order to homogenize and weigh the test material. Afterwards, the test material remained in a very viscous paste-like state. The test material could therefore not be crushed and ground using pestle and mortar and was treated like a paste. Prior to treatment the test material was heated again to 60.0 °C and placed on a nylon mesh. The test material was then applied directly on top of the skin tissue.

Application/Treatment of the Test Material
The test was performed on a total of 3 tissues per test material together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test material to the tissue and the solid test material was added in excess amount into 12-well plates on top of the skin tissues, using a nylon mesh. The test material was difficult to apply since it remained stuck to the mesh. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. Negative and positive controls were shared with parallel studies. All information pertaining to shared tissues are archived in the raw data. Since the test material reacted with the MTT medium, in addition three killed tissues treated with test material and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. Since the test material showed color interference in aqueous conditions, in addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated
with assay medium instead of MTT solution during the MTT assay. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. The test material was difficult to remove, as it remained stuck to the skin and the insert. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 ± 1 hours at 37°C.

Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours ± 5 minutes at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 69 hours. Extractants were moved to a 96-wells plate. In several wells the isopropanol was colored red. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

To ensure close contact of the test material to the tissue and the solid test material was added in excess amount into 12-well plates on top of the skin tissues, using a nylon mesh. The test material was difficult to apply since it remained stuck to the mesh. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 ± 1 hours at 37°C.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

Other effects / acceptance of results:

The test material was checked for color interference in aqueous conditions. Addition of the test material to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0014 and ≥4.0 (overflow), respectively. Therefore, it was concluded that the test material induced color interference.
Because a color change was observed in the presence of MTT it was concluded that the test
material interacted with the MTT endpoint.
The individual OD570 measurements are presented in any other information on results.
The mean absorption at 570 nm measured after treatment with the controls are presented in
any other information on results.
Table 2 (see any other information on results) shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the positive control compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test material, The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.9%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Table 4 in any other information on results) and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤1.5). The standard deviation value of the percentage viability of three tissues treated identically with positive and negative controls was ≤ 2.3%, indicating that the test system functioned properly.
During treatment with test material, it was noted that the test material was very viscous and was difficult to apply to the tissues, since it remained stuck to the nylon mesh. As a result, the tissues might not have been completely covered. In addition, the test material was difficult to remove from the tissues and inserts. As a result, any remaining test material colored the isopropanol red, which might have interfered with the measurements. These properties have interfered with the experiment, giving unreliable results. In addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. For one of the three tissues an ‘overflow’ was measured, which equals to an OD ≥ 4.0. As a result, the non-specific color by the test material would be ≥121% of the negative control tissues, which does exceed the acceptability criteria and the results. This high measurement, similar to in the test for color interference, is caused by residual test material, which also colored the isopropanol red. In addition to the normal procedure, three killed tissues treated with test material and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test material was -2.8% of the negative control tissues.
Since the %NSMTT was below 0.0, there was no need to correct for MTT interference. Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay. Since the %NSMTT was below 0.0, there was no need to correct for the non-specific color interference on killed tissues (NSCkilled).
Due to the properties of the test material, the high color interference, and the red-colored isopropanol, the relative mean tissue viability after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was not reliable and, therefore, not reported.

Any other information on results incl. tables

Table 1

Mean Absorption in the In Vitro Skin Irritation Test with Red HF2

 

	A	B	C	Mean		SD
	(OD570)	(OD570)	(OD570)	(OD570)
Negative control	1.139	1.088	1.116	1.114	±	0.026
Test item	(1)	(1)	(1)	(1)	±	(1)
Positive control	0.104	0.062	0.098	0.088	±	0.023

OD = optical density SD = Standard deviation

(1)  Not reported since the results are unreliable, due to the properties of the test material, high

%NSCliving (color interference), and red isopropanol after formazan extraction. Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.044). Isopropanol was used to measure the background absorption.

 

Table 2

Mean Tissue Viability in the In Vitro Skin Irritation Test with Red HF2

 

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	2.3
Test item	(1)	(1)
Positive control	7.9	2.1

(1)  Not reported since the results are unreliable, due to the properties of the test material, high

%NSCliving (color interference), and red isopropanol after formazan extraction.

Table 3

Individual OD Measurements at 570 nm

 

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570 measurement 1	1.1865	1.1390	1.1645
OD570 measurement 2	1.1792	1.1247	1.1547
Test material on viable tissue
OD570 measurement 1	0.2884 ^(1)	0.2511 ^(1)	1.0685
OD570 measurement 2	0.2837 ^(1)	0.2699 ^(1)	1.0566
Test material on viable tissue
(no MTT)
OD570 measurement 1	0.1255	0.0557	Overflow ^(1)
OD570 measurement 2	0.1257	0.0573	Overflow ^(1)
Non treated killed tissue
OD570 measurement 1	0.1133	0.1108	0.1186
OD570 measurement 2	0.1121	0.1069	0.1214
Treated killed tissue
OD570 measurement 1	0.0985	0.0682	0.0742
OD570 measurement 2	0.1000	0.0785	0.0737
Treated killed tissue (no MTT)
OD570 measurement 1	0.0854 ^(1)	0.0496	0.0866 ^(1)
OD570 measurement 2	0.0826 ^(1)	0.0489	0.0873 ^(1)
Positive control
OD570 measurement 1	0.1479	0.1038	0.1431
OD570 measurement 2	0.1481	0.1073	0.1409

⁽¹⁾ isopropanol was colored red after formazan extraction. OD = Optical density

Overflow = OD ≥ 4.0

Triplicate exposures are indicated by A, B and C.

Table 4

Historical Control Data for In Vitro Skin Irritation Studies

 

 

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Min	0.507	0.021
Max	1.478	0.549
Mean	1.090	0.097
SD	0.164	0.077
n	177	177

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 May 2019 to May 2022.

 

Applicant's summary and conclusion

Interpretation of results:

other: Inconclusive

Conclusions:

In conclusion, due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.

Executive summary:

The objective of this study was to evaluate Red HF2 for its ability to induce skin irritation on a human three-dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of the test material was tested through topical application for
15 minutes. 
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch 164:1 of the test material was a dark red solid. The test material was applied undiluted The solid test material liquified by heating until 65.0 °C in order to homogenize and weigh the test material. Afterwards, the test material remained in a paste-like state. Prior to treatment the test material was heated again to 60.0 °C and an excess amount was placed on a nylon mesh. Skin tissue was moistened with 5 μL of Milli-Q water and the test material was then applied directly on top of the skin tissue for 15 ± 0.5 minutes.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.9%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and
upper acceptance limit 1.5). The standard deviation value of the percentage viability of three tissues treated identically with positive and negative controls was ≤ 2.3%, indicating that the test system functioned properly.
During treatment with test material, it was noted that the test material was very viscous and was difficult to apply to the tissues, since it remained stuck to the nylon mesh. As a result, the tissues might not have been completely covered. In addition, the test material was difficult to remove from the tissues and inserts. As a result, any remaining test material colored the isopropanol red, which might have interfered with the measurements. These properties have interfered with the experiment, giving unreliable results.
In addition to the normal procedure, three tissues were treated with test material. Instead of MTT solution these tissues were incubated with assay medium. For one of the three tissues an ‘overflow’ was measured, which equals to an OD ≥ 4.0. As a result, the non-specific color by
the test material would be ≥121% of the negative control tissues, which does exceed the acceptability criteria and the results. This high measurement, similar to in the test for color interference, is caused by residual test material, which also colored the isopropanol red.
In addition to the normal procedure, three killed tissues treated with test material and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test material was -2.8% of the negative control tissues.
Since the %NSMTT was below 0.0, there was no need to correct for MTT interference.
Furthermore, since the test material was identified as MTT reducer and caused color interference, the test material was applied to three killed tissue replicates which underwent the entire testing procedure but were incubated with assay medium instead of MTT solution during the MTT assay. Since the %NSMTT was below 0.0, there was no need to correct for the non-specific color interference on killed tissues (NSCkilled).
Due to the properties of the test material, the high color interference, and the red-colored isopropanol, the relative mean tissue viability after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was not reliable and, therefore, not reported.
Results are retained within the raw data In conclusion, due to the properties of the test material, Red HF2 was deemed not fit for this in vitro skin irritation test.