Trisodium 3-[[4-[[6-(anilino)-1-hydroxy-3-sulphonato-2-naphthyl]azo]-5-methoxy-o-tolyl]azo]naphthalene-1,5-disulphonate

Endpoint summary

Description of key information

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. Additional controls were included to account for direct MTT interference and color interference. The relative mean viability of the test substance treated tissues was 102.6 % for the 3 minutes exposure and 96.3% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. Additional controls were included to account for direct MTT interference and color interference. The relative mean viability of the test substance treated tissues was 59.8 %. Therefore it can be concluded that the substance is not irritant to the skin.

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. Opacity was strongly increased, while permeability was slightly increased compared to the negative controls. The calculation of the In Vitro Irritancy Score showed that the substance is severely damaging to the eyes. As it could not be excluded that staining of the cornea could have influenced the opacity readings, semi-quantitative histopathological evaluation of the corneas from eyes exposed to the substance was performed. This investigation gave scores below the threshold for classification of this material as Eye Category 1. Therefore the conclusion of the study is that the substance is not considered severely damaging to the eyes.

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test (OECD 492).

The substance proved to be an MTT reducer and interfered with color. Therefore additional controls, viable tissues without MTT addition and freeze-killed tissues were included. The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 9.7% (threshold for irritancy:≤60%), consequently the test item was irritant to eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 November 2016 to 15 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,

Version / remarks:

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ Human Skin Mode

Cell source:

other: EpiDerm™ Human Skin Mode

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Human Skin Mode (MatTek)
- Tissue batch number(s):23385
- Delivery date: 13 December 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsed under a constant soft stream of DPBS and blotted dry with a tissue

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nM

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control and color interference
- Killed tissues: for MTT interference and correction for color interference
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 2/treatment
- Method of calculation used:
(TKT-UKT) + (CCT-CCU) – (ktCCT-ktCCU)
UKT Treated killed
TKT Untreated killed
CCT Color correction treated
CCU Color correction untreated
ktCCU Killed color correction treated
ktCCT Killed color correction untreated

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50% (H314 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (H314 1B or 1C)
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15% (not classified as corrosive)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: MTT reduction controls and killed color correction

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg as such

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 µL distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

The plates was incubated (37 ˚C, 5% CO2) for 3 hours in presence of MTT. Thereafter tissues were placed in isopropanol for MTT extraction, which was measured in triplicate samples as optical density at 562 nm.

Number of replicates:

2/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

A 3 minutes exposure

Value:

102.6

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

B 60 minutes exposure

Value:

96.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

The results were in agreement with the quality criteria:

Negative Control
The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

 

	Exposure time	Mean OD562	Relative mean viability
Negative control	3 min	1.694	100%
	60 min	1.773	100%
Test substance	3 min	1.783*	102.6%
	60 min	1.707**	96.3%
Positive control	3 min	0.058	3.4%
	60 min	0.051	2.9%

*Correction factor 0.020

** Correction factor 0.151

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered non-corrosive

Executive summary:

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. Additional controls were included to account for direct MTT interference and color interference. The relative mean viability of the test substance treated tissues was 102.6 % for the 3 minutes exposure and 96.3% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January 2017 to 20 February 2017

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:

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

Version / remarks:

amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s):17-EKIN-007 (0.38cm2)
- Delivery date: 14 February 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS:rinsed using a wash bottle containing DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nM

NUMBER OF REPLICATE TISSUES: 3/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control and color interference
- Killed tissues: for MTT interference and correction for color interference
- Procedure used to prepare the killed tissues (if applicable): water killed
- N. of replicates : 3/treatment
- Method of calculation used:
(TKT-UKT) + (CCT-CCU) – (ktCCT-ktCCU)
UKT Treated killed
TKT Untreated killed
CCT Color correction treated
CCU Color correction untreated
ktCCU Killed color correction treated
ktCCT Killed color correction untreated

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Relative mean tissue viability is ≤50% --> irritant (H315)
- Relative mean tissue viability is >50% --> non-irritant (not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: MTT reduction controls and killed color correction

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg as such ( (26.3 mg/cm2)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL SDS (5%)

Duration of treatment / exposure:

15 min at room temperature (thereafter rinsed with DPBS

Duration of post-treatment incubation (if applicable):

The plates were incubated (37 ˚C, 5% CO2) for 42 hours thereafter shaken to homogenize. Thereafter incubated for 3 hours in presence of MTT. The epidermis and the collagen matrix were separated and immersed in acidified isopropanol. The tissues were stored in a refrigirator for 6 days at 1-10 ˚C for MTT extraction, which was measured in triplicate samples as optical density at 562 nm.

Number of replicates:

3/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

59.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-irritant

Other effects / acceptance of results:

The results were in agreement with the quality criteria:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

   

	Exposure time	Mean OD562	Relative mean viability	SD
Negative control	15 min	0.704	100%	10.9%
Test substance	15 min	0.421	59.8%	5.7%
Positive control	15 min	0.095	13.5%	4.9%

*Correction factor applied 0.406

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered non-irrirant

Executive summary:

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. Additional controls were included to account for direct MTT interference and color interference. The relative mean viability of the test substance treated tissues was 59.8 %. Therefore it can be concluded that the substance is not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 (histopathology 5 June 2017 to 16 June 2017)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Principles of method if other than guideline:

High opacity scores were obtained in the test and the corneas were stained dark purple by the substance, and thus, the opacity scores may have been due to the staining. Therefore conform the guidance additional histopathology was performed

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not indicated
- Storage, temperature and transport conditions of ocular tissue: placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), transported over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: not indicated
- indication of any existing defects or lesions in ocular tissue samples: checked and only undamaged tissue used

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 20% w/v solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes at 32 ± 1 ºC

Duration of post- treatment incubation (in vitro):

post treatment opacity measurement immediately
permeality measurement after exposure to fluorescein for 90 min at 32 ± 1 ºC

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS:based on visual examination and pre-treatment opacity check

NUMBER OF REPLICATES: 2/treatment

TREATMENT METHOD:closed chamber

REMOVAL OF TEST SUBSTANCE: 3 rinses with EMEM containing phenol red

MEASURED ENDPOINTS: Corneal opacity and Corneal permeability

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

Histologically process and microscopically evaluation of the isolated bovine eye corneas was performed after in vitro testing.

Irritation parameter:

in vitro irritation score

Value:

138.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

It can not be excluded that staining of the cornea could have influenced the opacity readings

Irritation parameter:

histopathological observations

Value:

>= 1 - <= 2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

indication of irritation is not sufficient to allow the conclusion severely damaging to the eyes

Other effects / acceptance of results:

The positive control was slightly out of range. This was evaluated and considered not to have affected the study validity.

The purple staining of cornea by the test substance may have influenced the opacity reading. This was checked by histologically processing and microscopically evaluation of the isolated bovine eye corneas after in vitro testing (see below)

The histopathology findings were only low grade and restricted to the upper layers of the epithelium, so were not typical of the histopathological findings that would be expected following exposure of the cornea to substances having the potential to cause serious eye damage associated with dense corneal opacity (i.e. not consistent with the corneal effects commonly induced by Category 1 substances). So, it is very likely that the high scores for opacity in the BCOP test were due primarily to staining of the cornea by the test item.

	Opacity		Permeability
Cornea	Post-Treatment- Pre-Treatment	Corrected Value	OD	Corrected Value	In Vitro Irritancy Score
Negative control	0		0.029
	1		0.022
	1		0.054
Mean	0.7*		0.035		1.2
Positive Control	83	82.3	1.239	1.204
	84	83.3	1.609	1.574
	82	81.3	1.482	1.447
Mean		82.3		1.408	103.5
Test Item	182	181.3	0.230	0.195
	135	134.3	0.043	0.008
	94	93.3	0.243	0.208
Mean		136.3		0.137	138.4

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of the test the substance is considered not severely damaging to the eyes

Executive summary:

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. Opacity was strongly increased, while permeability was slightly increased compared to the negative controls. The calculation of the In Vitro Irritancy Score showed that the substance is severely damaging to the eyes. As it could not be excluded that staining of the cornea could have influenced the opacity readings, semi-quantitative histopathological evaluation of the corneas from eyes exposed to the substance was performed. This investigation gave scores below the threshold for classification of this material as Eye Category 1. Therefore the conclusion of the study is that the substance is not considered severely damaging to the eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2018 to 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

9th October 2017
MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of
acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human Ep
iOcular™ Model; 29 June 2015.

GLP compliance:

yes (incl. QA statement)

Species:

other: human keratinocytes

Details on test animals or tissues and environmental conditions:

-EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).
Lot No.: 27027
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:1 mg/mL MTT
- Incubation time: 3 h
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1)

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control and color interference
- Freeze killed tissues: for MTT interference and correction for color interference

METHOD OF CALCULATION
True viability [%] = TI viability – mean TestItemKC Viability – mean TestItemCC Viability + mean TestItemNSKC viability

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment

PREDICTION MODEL / DECISION CRITERIA
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant.

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes immersion incubation (post-soak to remove any substance or control absorbed ) followed by post-treatment incubation for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2.

Number of animals or in vitro replicates:

2/treatment

Details on study design:

At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).
After rinsing (that did not remove the substance completely), the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 minutes immersion incubation (post-soak) at room temperature.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for 2 to 3 hours at room temperature). For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Irritation parameter:

other: viability %

Run / experiment:

1

Value:

9.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.038	0.036	0.037
Negative Control	1	1.760	1.714	1.737	1.700	1.711	99.4	1.3	100.0
	2	1.781	1.736	1.759	1.722		100.6
Positive Control	1	0.632	0.609	0.621	0.584	0.530	34.1	6.4	30.9
	2	0.525	0.499	0.512	0.475		27.8
Test Item	1	0.220	0.210	0.215	0.178	0.178	10.4	0.0	9.7***
	2	0.218	0.212	0.215	0.178		10.4
Blank		0.038	0.036	0.037
Negative Control Viable Tissues	1	0.043	0.043	0.043	0.006	0.006	0.3	0.0	0.4
	2	0.044	0.043	0.043	0.006		0.4
Test Item Viable Tissues	1	0.059	0.053	0.056	0.019	0.013	1.1	0.7	0.8
	2	0.045	0.044	0.045	0.008		0.4
Negative Control Freeze killed Tissues	1	0.090	0.089	0.090	0.053	0.045	3.1	0.9	2.6
	2	0.073	0.074	0.073	0.037		2.1
Test Item Freeze killed Tissues	1	0.084	0.083	0.084	0.047	0.043	2.7	0.4	2.5
	2	0.077	0.076	0.076	0.039		2.3

* Relative viability [rounded values]: 100 * (absorbance test item/positive control)/negative control)/absorbance negative control

**Mean relative viability [rounded values]: 100 * (mean absorbace test item/ control/negative control)/mean absorbance negative control

*** corrected value True viability [%]: mean absorbance_{test item}– mean absorbance_{additional viable tissue test item treated}– (mean absorbance_{test item treated killed control}– mean absorbance_{negative control of freeze killed tissues})

 

 

Interpretation of results:

other: irritant to the eyes

Conclusions:

The substance is irritant to the eyes

Executive summary:

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test.

The substance proved to be an MTT reducer and interfered with color. Therefore additional controls, viable tissues without MTT addition and freeze-killed tissues were included.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 9.7% (threshold for irritancy:≤60%), consequently the test item was irritant to eye.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The outcome of the BCOP test based on the histopathology performed is that the substance is not severely irritating to the eyes. Therefore a study according to OECD 492 was initiated which shows that the substance is irritating to the eyes. In a weight of evidence this information will lead to a classification as eye irritant (category 2).

Justification for classification or non-classification

Based on the results of the available studies, it can be concluded that the substance does not need to be classified for irritation to skin according to Regulation (EC) No 1272/2008 (CLP), but is considered an eye-irritant and needs to be classified as H319 (category 2).