Disodium 4-hydroxy-3-[[2-methoxy-5-methyl-4-[(4-sulphonatophenyl)azo]phenyl]azo]-7-(phenylamino)naphthalene-2-sulphonate

Endpoint summary

Description of key information

Not irritating for skin

Irritating for the eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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 system:

human skin model

Remarks:

EPISKIN Reconstructed Human Epidermis Model Kit

Cell type:

other: reconstructed (RhE)

Details on animal used as source of test system:

The procedure followed is based on the recommended EpiSkin SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study

Vehicle:

unchanged (no vehicle)

Details on test system:

A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tested substance - mean of replicates

Value:

117.53

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control group - mean of replicates

Value:

100

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control group - mean of replicates

Value:

13.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into blue formazan salt that is quantified after extraction from tissues (Mosman T., 1983). The reduction of cell viability in treated tissues is compared to negative controls and expressed as a percentage. The percentage reduction in viability is used to predict the irritation potential.

Each  test  substance  (test  material,  negative  and  positive  controls)  is  topically  applied concurrently  on  three  tissues replicates  for  42 minutes  at  room temperature  (RT,  comprised between  18°C  to  24°C).  Exposure  to  the  test  substance  was  followed  by  rinsing  with phosphate  buffer  saline  (PBS)  and mechanically dried. Epidermis were then transferred to fresh  medium  and  incubated  at  37°C  for  42  additional  hours.  Cell  viability  is  assessed  by incubating  the  tissues  for  3  hours  with  0.3  mL MTT  solution  (1  mg/mL). The  formazan crystals are extracted  using 1.5  mL  isopropanol  for  2  hours  at  RT  and  quantified  by spectrophotometry  at  570  nm wavelength. Sodium  Dodecyl Sulphate (SDS 5%), and PBS treated  epidermis  are  used  as  positive  and  negative  controls, respectively. For each treated tissue,  the  cell  viability  is  expressed  as  the  percentage  of  the  mean  negative  control  tissues. Values under 50% is qualified the test substance as irritant.

Interpretation of results:

GHS criteria not met

Conclusions:

Not irritant for skin

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 %. The test results does not show any effect on skin. The tested substance could be considered as not irritant for the skin and does not required any classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

in vitro

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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

Vehicle 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 infl uenced the opacity readings

Irritation parameter:

histopathological observations

Value:

>= 1 - <= 2

Vehicle 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.
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.

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:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The substance is not considered severely damaging to the eyes but it's considered as eye irritant.

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.

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

Skin irritation:

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.

Eye irritation:

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 and needs to be classified as H319.