Xanthylium, 3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, molybdatetungstatephosphate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 13 September 2017 and 02 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Lumière Pink PTM 0245F
CAS Number: 1326-04-1
EC Number: 215-414-9
Batch: 280PE310516
Purity: UVCB (treat as 100%)
Physical state/Appearance: Dark magenta powder
Expiry Date: 01 July 2022
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

not specified

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 26 September 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-039
Maintenance Medium lot number : 17-MAIN3-041
Assay Medium lot number : 17-ESSC-038

Justification for test system used:

The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water killed tissues for quantitative correction of the results.
An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze killed tissues was performed. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and water killed tissues.
This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Water-killed tissues were prepared prior to the study by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 C, 5% CO2 in air for a minimum of 48 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (14 to 30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to three water killed tissues. In addition, three water killed tissues remained untreated. The untreated water killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 mg of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
The test item was found to produce a colored solution which may interfere with the MTT endpoint. Therefore, color correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for 3 hours post exposure instead of MTT. Three tissues were dosed with the test item and three remained untreated to act as negative controls.

Double Correction Check
A third set of controls was also used to prevent a double correction from a colored test item that also reduces MTT using killed tissues.
Intrinsically colored test items may bind to both living and killed tissues and therefore the non viable water killed tissues may not only correct for potential direct MTT reduction by the test item, but also for color interference arising from the binding of the test item to the killed tissues. This could lead to a double correction for color interference since the viable color interference tissues already corrects for color interference arising from the binding of the test item to living tissues.
Three water killed tissues were dosed with the test item and three water killed tissues remained untreated to act as the negative control. These tissues were incubated with assay medium instead of MTT post exposure.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:

Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre labeled 12 well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37”C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7 Minute contact time the SDS solution was re spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37”C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42 Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer at 14 to 30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3 Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT 4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

> 90.7 - < 95.6

Negative controls validity:

valid

Remarks:

Set to 100%

Positive controls validity:

valid

Remarks:

18.0%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

6.5 Quality Criteria
The test was repeated twice due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 18.0% relative to the negative control treated tissues and the standard deviation value of the viability was 5.5%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.939 and the standard deviation value of the viability was 5.3%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.5%. The test item acceptance criterion was therefore satisfied.

 Direct MTT Reduction

An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item. Therefore, an additional procedure using water killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water killed tissues for quantitative correction of results or for reporting purposes.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a dark purple color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that negligible color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.

 Double Correction Check

The results of the color correction tissues were not used; therefore it was unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₇₀values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table1.

The relative mean viability of the test item treated tissues was93.1% after a 15‑Minute exposure period and 42‑Hour post‑exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal

 

TABLE 1 Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.994	0.939	0.050	105.9	100*	5.3
	0.897			95.5
	0.927			98.7
Positive Control Item	0.138	0.169	0.051	14.7	18.0	5.5
	0.140			14.9
	0.228			24.3
Test Item	0.898	0.874	0.023	95.6	93.1	2.5
	0.852			90.7
	0.871			92.8

OD= Optical Density

SD=        Standard deviation

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

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to have the potential to cause color interference and therefore additional tissues were incorporated for color correction purposes. An assessment of the test item’s capability to directly reduce MTT was inconclusive due to the intrinsic color of the test item and therefore, an additional procedure using freeze‑killed tissues was performed. A third set of controls were included, comprising freeze‑killed tissues, in order to prevent a double correction from a colored test item that also reduces MTT. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was93.1% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period.

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

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation, other

Remarks:

the chorioallantoic membrane (CAM) of chicken eggswas investigated

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2004 - 23 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

The study procedures described in this report are based on the following papers:

Weterings, P.J.J.M. and van Erp, Y.H.M. (1987). Alternative methods in Toxicology Vol. V.: In Vitro Toxicology - Approaches to Validation. Ed. A.M. Goldberg, M.A. Liebert Inc. NY, USA.

LOpke, N.P. (1985), Hen's egg chorioallantoic membrane test for irritation potential, Fd. Chem. Toxicol. 23, 287.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:0245N/041020
- Expiration date of the lot/batch: 16 March 2005
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark

Species:

other: chicken egg

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: chicken breeding centre (Het Anker, Ochten, the Netherlands).
- Acclimation period:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.2mL

Duration of treatment / exposure:

20 seconds

Duration of post- treatment incubation (in vitro):

The effects on the blood capillaries were evaluated 30 seconds after application (immediately after rinsing) and 1.5 and 4.5 minutes later

Number of animals or in vitro replicates:

4 test eggs and 12 control eggs

Details on study design:

White eggs were incubated in the incubator with the air chamber upwards. The temperature was maintained at 37 ± 1°C and the relative humidity between 50 and 80%. The eggs were turned once every hour. They were candled at the seventh day of incubation; unfertilised eggs or eggs containing a dead embryo were removed.
On the tenth day of incubation the egg shell was opened above the air chamber with a rotating polisher, and the shell with the attached membrane was removed until the margin of the air chamber. The inner egg membrane was then carefully eliminated to expose the CAM. To avoid capillary bleeding, 0.1 ml of water was placed on a small nick made in the inner egg membrane before picking it up with a fine-pointed forceps. The test substance was applied to the CAM. Twenty seconds after application the CAM was carefully rinsed with approximately 7 ml of tepid water.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: 30 seconds, 2 mins, 5 mins

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 30 seconds, 2 mins, 5 mins

Score:

0

Max. score:

0

Reversibility:

other: n/a

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 30 seconds, 2 mins, 5 mins

Score:

0

Max. score:

0

Reversibility:

other: not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

other: 30 seconds, 2 mins, 5 mins

Score:

0

Max. score:

0

Reversibility:

other: not aapplicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

CAM ASSAY
Injection: No injection was observed.
Haemorrhage: No haemorrhages were observed.
Coagulation: No coagulation was observed.

LUMIERE PINK 0245N revealed neither corneal damage nor any effects on the CAM.

Interpretation of results:

GHS criteria not met

Conclusions:

LUMIERE PINK 0245N revealed neither corneal damage nor any effects on the CAM.

It is concluded that negligible irritation may be expected after acute eye exposure to test substance LUMIERE PINK 0245N.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2004 - 23 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

The study procedures described in this report are based on the following papers:

Weterings, P.J.J.M. and van Erp, Y.H.M. (1987). Alternative methods in Toxicology Vol. V.: In Vitro Toxicology - Approaches to Validation. Ed. A.M. Goldberg, M.A. Liebert Inc. NY, USA.

LOpke, N.P. (1985), Hen's egg chorioallantoic membrane test for irritation potential, Fd. Chem. Toxicol. 23, 287.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:0245N/041020, Source Sponsor
- Expiration date of the lot/batch: 16 March 2005
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: slaughterhouse (Vitelco, 's Hertogenbosch, the Netherlands)
- Acclimation period: kept in the humid atmosphere for approximately 10 minutes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): (approximately 20°C) under saline

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.1 ml/eye.

Duration of treatment / exposure:

0.5 minutes.

Duration of post- treatment incubation (in vitro):

10 minutes.

Number of animals or in vitro replicates:

5

Details on study design:

Bovine eyes were examined for injuries. This was done by holding the eyes submersed in saline; eyes with corneal damage or other abnormalities were discarded. The selected eyes were preserved at ambient temperature (approximately 20°C) under saline.
Total number of eyes: 5 eyes + 3 control eyes

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

morphological effects

Run / experiment:

Detachment

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Epithelial integrity

Remarks:

degree of staining

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance revealed neither corneal damage.
It is concluded that negligible irritation may be expected after acute eye exposure to test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available