Sodium 6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate

Endpoint summary

Description of key information

Skin irritation in vitro: not irritant

OECD 439: negative

Skin irritation in vivo: insufficient for classification according to CLP (UN/GHS)

Eye irritation in vitro: irritant Cat 2

OECD 437: inconclusive, not cat 1

OECD 492: cat 1 or 2

Eye irritation in vivo: insufficient for classification according to CLP (UN/GHS)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28, 2017 - August 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied neat to the tissues.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 17-RHE-083
- Expires: August 7, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: 0 - 3.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Acceptance Criteria Result
Cell viability (570 nm optical density, MTT test) OD >0.7 OD 1.2 (CV = 5.5 %)

Barrier function integrity test
(Exposure time inducing 50%
viability using Triton X-100 1%) 4.0h ≤ ET50 ≤ 10.0h 4.4h

Histological observation
(HES stained vertical paraffin sections) Number of cell layers ≥ 4 5.5 cell layers
Absence of significant
histological abnormalities. Absence of significant histological abnormalities.

Well differentiated epidermis Satisfactory
consisting of basal, spinous,
granular layers and a stratum c
orneum.


NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA:
A test item is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is ≤ 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

88.8

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 2.065 to 2.242

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 2.140
Mean viability positive control < 40% 1.3%
SD of group-mean value ≤ 18% 7.7% (positive control)
4.3% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 2.140
Mean viability positive control ≤ 2.97% 1.3%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 3.4%

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	2.140	100
Positive Control	42	0.028	1.3
Test Material	42	1.846	88.8

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-08-11 to 1981-08-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Only a brief summary of the study is available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

Appearance: yellow crystals

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before application, the test material was moistened with 0.4 mL 0.9% NaCl solution

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Hoechst AG, Frankfurt, Germany
-Weight at study initiation: 1.9- 2.7 kg
-Housing: single
-Diet: ad libitum
-Water: ad libitum

Type of coverage:

occlusive

Preparation of test site:

other: clipped and scarified

Vehicle:

physiological saline

Controls:

not required

Amount / concentration applied:

0.5 g per animal

Duration of treatment / exposure:

24 hours

Observation period:

up to 8 days

Number of animals:

6

Details on study design:

TEST SITE
-Area of exposure: 6*3 cm on the flank
-Type of wrap if used: adhesive plaster (Hansamed) and polyethylene foil

REMOVAL OF TEST SUBSTANCE
-Method: not specified
-Time after start of exposure: 24 hours

OBSERVATION TIME POINTS
-After removal of the plaster and after 48 and 72 hours
d then daily up to experimental day 8

SCORING SYSTEM
-According to OECD GL 404

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: not further specified

Score:

1.6

Reversibility:

not specified

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

In two rabbits effects on the skin (dry, rough, cracked, eschar formation) was noted

Study design

In this study performed equivalent to OECD GL 404, 500 mg of the test item was mixed with physiological saline soution to ensure good contact to the skin. Afterwards, the test material was applied to clipped and scarified skin for a 24 hours period under occlusive conditions. Scoring was performed after pathch removal (24 h), and after 48, and 72 hours.

Results

Under the conditions of the present study, the treatment caused a mild irritation (mean score: 1.6).

Conclusion

A mild irritating potential could be detected.

Interpretation of results:

study cannot be used for classification

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Only a brief summary of the study is available

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

Description: yellow crystals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
-Source: Hoechst AG, Frankfurt, Germany
-Weight at study initiation: 1.9 - 2.7 kg
-Housing: groupwise
-Diet: ad libitum
-Water: ad libitum

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

100 mg test item moistened with 0.04 mL physiological saline solution

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

up to 72 hrs

Number of animals or in vitro replicates:

6

Details on study design:

A volume of 100 mg of the test material was instilled into the conjunctival sac of the left eye, while the right eye remained untreated and was used for control purposes.

REMOVAL OF TEST SUBSTANCE
-The eyes were rinsed 24 hrs after application.

SCORING SYSTEM: according to OECD GL 405

The 48/72 hrs score was obtained after installation of one drop of a 0.01% fluorescein sodium.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 7 hours

Score:

6

Reversibility:

not specified

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test material gave no indication for an eye irritation potential in rabbits.

Executive summary:

This study was performed equivalent to OECD GL 405 and gave no indication for an eye irritation potential in rabbits.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28, 2017 - August 14, 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)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaption to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

PREPARATION OF THE TEST MATERIAL
The test item was prepared as a 20% (w/v) solution in a 0.9% sodium chloride solution. The stability in the vehicle was not investigated. The test item preparation was administered within <1 hour after preparation.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 750 µL (i.e. 150mg/750µL)

NEGATIVE / VEHICLE CONTROL: 750 µL

Designation: 0.9% sodium chloride solution
Supplier: B. Braun Melsungen AG, Germany
Batch: 16435011
Storage: 2 to 8°C
Released until: September 2019


POSITIVE CONTROL: 750 µL

Designation: Art. 814223
Synonym: Imidazole
Supplier: Merck KGaA, Germany
Batch: S6746923
Purity (GC) 99.8% (a/a)
Storage: At room temperature
Released until: August 31, 2018

Imidazole was dissolved with 0.9% sodium chloride solution to a concentration of 20% (w/v).

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

in vitro: triplicate design

Irritation parameter:

in vitro irritation score

Run / experiment:

Run 1 / Experiment 1

Value:

3.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

According to OECD 437 no prediction can be made regarding the eye hazard potential of the test item.

Other effects / acceptance of results:

OTHER EFFECTS:
- All corneas treated with the test item preparation showed a slight yellowish stain after removing the test item preparation by washing with wash medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.2 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 – 3.2).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 106.4 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 80.5 – 133.0).

Therefore, the study fulfilled the acceptance criteria.

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	SD
Negative control	0.9% NaCl Solution	0.0	0.002	0.030	0.2	0.4
		0.0	0.003	0.045
		0.6	0.004	0.660
Positive control	20% Imidazole solution	71.0	2.506	108.590	106.4	2.3
		71.2	2.352	106.480
		72.4	2.112	104.080
Test item	Test item	4.7	0.017	4.955	3.4	1.5
		2.8	0.015	3.025
		1.8	0.018	2.070

Interpretation of results:

other: Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Conclusions:

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 17, 2017 - November 8, 2017

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:

October 9, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,

Version / remarks:

September 14, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No pre-treatment, the test item was applied neat to the tissues.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 50 mg per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: MatTek In Vitro Life Science Laboratories
Lot-No.: 032817ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: March 28, 2018
Storage: 15 to 30°C

Duration of treatment / exposure:

6 hours

Number of animals or in vitro replicates:

in vitro: duplicate design

Irritation parameter:

other: Viability %

Run / experiment:

Run 1 / Experiment 1

Value:

4.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

According to OECD 492 the test item is labeled irritant (UN GHS Category 1 or Category 2).

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.184 and 1.186).
2. The mean relative viability of the positive control is below 50% of the negative control viability (17.3%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.1% to 1.5%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

	Mean OD	Mean Viability
Negative Control	1.185	100.0%
Positive Control	0.204	17.3%
Test Item	0.052	4.4%

Interpretation of results:

other: The test item is labeled irritant (UN GHS Category 1 or Category 2).

Conclusions:

Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 492. Under the conditions of the present study, the test item did show an eye hazard potential. The test item is labeled irritant (UN GHS Category 1 or Category 2).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification for skin irritation according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.

Based on the OECD 437 result eye damage cat 1 can be excluded. Taking all test results into account there is need for classification for eye irritation category 2 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776. Based on Test