1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]bisanthraquinone

Endpoint summary

Description of key information

The test article is not irritating to the skin and not irritating to the eye as observed in in vitro studies.

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:

31 Oct 2014 - 02 Feb 2015

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)

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 640/2012 of 6 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) Nº 440/2008, B.46 B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

Three dimensional human epidermis model EpiDermTM

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Amount/concentration applied:

25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. A nylon mesh was placed carefully onto the tissue surface afterwards.

Duration of treatment / exposure:

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator (1h exposure total).

Number of replicates:

Three tissues were treated with the test substance, the PC and NC, respectively.

Controls:

other: Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC).

Details on study design:

The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 tissues

Run / experiment:

#1

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test substance is not able to reduce MTT directly, as determined in a pretest.

Individual and mean OD570 values, individual and mean viability values and standard deviations

substance		tissue 1	tissue 2	tissue 3	mean	SD
NC	mean OD570	2.809	2.714	2.843	2.789	0.067
	viability [% of NC]	100.7	97.3	101.9	100	2.4
test substance	mean OD570	2.931	2.561	2.979	2.824	0.229
	viability [% of NC]	105.1	91.8	106.8	101	8.2
PC	mean OD570	0.101	0.089	0.115	0.101	0.013
	viability [% of NC]	3.6	3.2	4.1	4	0.5

Interpretation of results:

GHS criteria not met

Conclusions:

The test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Executive summary:

The potential of the test article to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 14 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101%. Based on the observed results it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

other: in vitro turn key strategy

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct 2014 - 02 Feb 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD (2014a) Draft Proposal for a New Test Guideline: Reconstructed Human Cornealike Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage

Principles of method if other than guideline:

The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three dimensional human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue the induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol-extraction of the formazan from the tissues, the optical density of the extract is determined spectrophotometrically. Optical density of the extracts of test-substance treated tissues is compared to values from negative control tissues and expressed as relative tissue viability.

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed three dimensional human cornea model

Strain:

other: Tissue model: OCL-200

Details on test animals or tissues and environmental conditions:

The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on especially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control (NC): De-ionized water, sterile Positive control (PC): Methyl acetate (98+%, CAS No.: 79-20-9)

Amount / concentration applied:

TEST MATERIAL
The test substance is applied undiluted; therefore no preparation of the test substance in a vehicle was performed.

Duration of treatment / exposure:

After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Number of animals or in vitro replicates:

Two tissues were treated with each, the test substance, the PC and the NC.

Details on study design:

PREINCUBATION OF THE TISSUES
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

PRETREATMENT OF THE TISSUES
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

APPLICATION OF THE TEST SUBSTANCE
Using a sharp spoon, a bulk volume of 50 μL of the test material was applied covering the whole tissue surface.
Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC).
After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

REMOVAL OF TEST SUBSTANCE
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed
medium (post-soak immersion) in order to remove residual test substance. After 25 minutes (solids) of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).

Irritation parameter:

other: Tissue viability in %

Remarks:

average of 2 samples

Run / experiment:

#1

Value:

104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects:

The test substance is not able to reduce MTT directly.

RESULTS OF THE EPIOCULAR TEST

Test substance		tissue 1	tissue 2	mean	inter-tissue variability [%]
NC	mean OD570	1.555	1.424	1.490
	viability [% of NC]	104.4	95.6	100	8.8
14/0579-1	mean OD570	1.687	1.410	1.548
	viability [% of NC]	113.2	94.7	104	18.6
PC	mean OD570	0.400	0.363	0.382
	viability [% of NC]	26.9	24.4	26	2.5

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results for the EpiOcular Test it was concluded, that the test article does not show an eye irritation potential under the test conditions chosen.

Executive summary:

The potential of the test article to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 20 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by a 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results:

The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 104%.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The potential for corrosive activity and skin irritation of the test article was assessed in vitro. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). The potential of the test item to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 6 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT), was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 112%, and it was 106% after an exposure period of 1 hour (SCT). The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 101% (SIT). Based on the observed results and applying the evaluation criteria it was concluded, that the test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation

The potential for ocular irritation of the test article was assessed in vitro. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test. However, in the current case for the test item the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived. The potential of the test item to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 12 mg) of the undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT), was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The EpiOcular™ eye irritation test showed the following results: The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 104%. Based on the observed results for the EpiOcular Test alone it was concluded, that the test item does not show an eye irritation potential under the test conditions chosen.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the substance is not considered to be classified for irritation under Regulation (EC) No 1272/2008.