1,2-Benzenediol, 4-[(4-methylphenyl)sulfonyl]-

Endpoint summary

Description of key information

No indication was found for irritation or corrosion effects on the skin or eyes.

For skin irritation/corrosion, two in vitro tests were negative.

For eye irritation, an in vitro test was not sufficient to decide on eye irritation properties. Therefore, a read-across from a structural analogue was used to help exclude any eye irritation effects.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): 14-EKIN-003
- Delivery date: 04 February 2014
- Date of initiation of testing: 05 February 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml of 0.3 mg/ml MTT solution
- Incubation time: 3 hours +- 5 minutes
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: not reported
- Filter bandwidth: not reported
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
not reported

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not needed

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive Cat 1A to skin if the viability after 3 minutes exposure is less than 35%,
- The test substance is considered to be corrosive Cat 1B or 1C if the viability after 3 minutes exposure is greater than or equal to 35 % and the viability after 1 hour exposure is less than 35%, or if the viability after 1 hour exposure is greater than or equal to 35% and the viability after 240 minutes exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 240 minutes exposure is greater than or equal to 35% .

Amount/concentration applied:

20 mg of test item was applied topically to the corresponding tissues and 100 µL of 0.9% w/v sodium chloride solution was added for wetting the test item.

Duration of treatment / exposure:

3, 60 and 240 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Preparation of test site:

other: not applicable

Duration of treatment / exposure:

/

Observation period:

/

Number of animals:

/

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes exposure

Value:

102

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

79.2

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

93.8

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Following the in-vitro OECD 431 test, the substance is not skin corrosive according to CLP criteria.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

The EPISKIN model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. 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 (OD) was measured at 562 nm (OD562).

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 viabilities of the test item treated tissues were:

240 minutes exposure: 102.0%
60 minutes exposure: 79.2%
3 minutes exposure: 93.8%

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

Conclusion

The test item was classified as non-corrosive to the skin. The following classification criteria apply:
EU DSD (67/548/EEC): Not classified for corrosivity.

EU CLP (1272/2008/EC)/UN GHS: Not classified for corrosivity.

UN Packing Group: Non-Corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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)

Principles of method if other than guideline:

/

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Reconstructed Human Epidermis Model
- Tissue batch number(s): 14-EKIN-006
- Production date: /
- Shipping date: /
- Delivery date: 25 February 2014
- Date of initiation of testing: 25 February 2014 (pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml of 0.3 mg/ml MTT
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: /
- Filter bandwidth: /
- Linear OD range of spectrophotometer: /

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3


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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if relative mean tissue viability is <= 50%.
- The test substance is considered to be non-irritant to skin if relative mean tissue viability is >50%
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Triplicate tissues were treated with test tiem for 15 min. Test item was applied topically to the tissues.
5 microL of sterile distilled water was applied to the epidermal surface. 10 mg of the test item was then applied to the epidermal surface.
Triplicate tissues treated with 10 microL of DPBS served as negative controls.
Triplicate tissues treated with 10 microL of SDS 5% served as positive controls.

Duration of treatment / exposure:

15 min, then rinsed. Rinsed tisseus were incubated at 37°C, 5% CO2 in air for 42 hours.
Then shaked for 15 min.
2 mL of 0.3 mg/mL MTT solution pipetted into the third columnd of 3 wells of the 12- well plate(s). Incubated for 3h at 37°C, 5% CO2 in air.

Irritation / corrosion parameter:

% tissue viability

Value:

>= 72.4 - <= 92.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
The relative mean viability of the test ite treated tissues was 82.1% after a 15-minute exposure period and 42 hours post-exposure incubation period.

The relative mean tissue viability for the positive control tissues was 7.1% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.7%. The positive control acceptance criterion was therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.959 and the standard deviation value of the percentage viability was 8.6%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically test item treated tissues was 10.1%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance was classified as non-irritant according to CLP criteria.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed 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. The concentration of the inflammatory mediator IL-la in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

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. 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 562 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 was 82.1% 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 DSD & 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: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Principles of method if other than guideline:

None.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

The 2 animals received an acclimatisation period of at least five days. The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. The rate of air exchange wat at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve houds continuous light (06:00 ti 18:00) and twelve hours darkness. the animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: left eye of the animal used as control

Amount / concentration applied:

0.1 mL of test item (94 mg) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

Duration of treatment / exposure:

Eyelids were held together for about one second imeediately after treatment, to prevent loss of the test item, and then released.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

2

Details on study design:

Assessment of ocular damage/irritation was made approx. 1 hour, 24, 48 and 72 hours following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

3.3

Max. score:

7

Reversibility:

fully reversible within: 72h

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.833

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.5

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: conjunctivae: discharge

Basis:

mean

Time point:

24/48/72 h

Score:

0.333

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24h observation with minimal conjunctivak irritation noted in both treated eyes at the 48h observation.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to CLP.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

Results

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72 -hour observation.

Conclusion

The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to CLP.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

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:

None.

GLP compliance:

yes (incl. QA statement)

Species:

other: freshly slaughtered cattle

Details on test animals or tissues and environmental conditions:

The eyes were excised by an abattoir employee and places in HBSS (Hanks' Balanced Salt Solution), supplemented with Penicilin and Streptomycin, and transported on ice packs. The corneas were prepared immediately on arrival.

Vehicle:

other: test item was prepared as a 20 w/v solution in 0.9%w/v sodium chloride solution

Controls:

other: control corneas

Amount / concentration applied:

0.75 mL of the test item preparation (or control items) were applied to the appropriate corneas.

Duration of treatment / exposure:

Each holder was incubated at 32+-1 °C for 240 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

3 corneas for test item. 3 for negative and 3 for positive control.

Details on study design:

Opacitiy and permeability were tested. Opacity was measured before and after test.
Permeability was measured with sodium fluorescein.

Irritation parameter:

in vitro irritation score

Value:

30.9

Vehicle controls validity:

valid

Remarks:

IVIS = 73

Negative controls validity:

valid

Remarks:

IVIS = 1.9

Interpretation of results:

other: not corrosive

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.
The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

See attached documents.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
See attached documents.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
See attached documents.

4. DATA MATRIX
See attached documents.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Principles of method if other than guideline:

None.

GLP compliance:

yes (incl. QA statement)

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

3.3

Max. score:

7

Reversibility:

fully reversible within: 72h

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0.833

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.5

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: conjunctivae: discharge

Basis:

mean

Time point:

24/48/72 h

Score:

0.333

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24h observation with minimal conjunctivak irritation noted in both treated eyes at the 48h observation.

Interpretation of results:

GHS criteria not met

Conclusions:

Read-across from CH03951 was used to determine the eye irritation potential of CH04008.
The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available