Registration Dossier

Diss Factsheets

Administrative data

Description of key information

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:
The study was performed between 02 September 2009 and 07 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
other: EU Guideline Testing of Chemicals B46
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Draft Test Guideline (version 4)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspectection: 19-08-2009 Date of Signature: 04-03-2009
Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals or test system and environmental conditions:
Not applicable
Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

- The test Material was applied neat.

- Amount(s) applied (volume or weight with unit):
10µl of of the test material was applied to the epidermis surface.

- Concentration (if solution):
The test material was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
15 Minutes & 42 hour post exposure incubation
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
10µl of the test material was applied to the epidermis surface.

- % coverage:
The test material was applied topically to the corresponding tissues ensuring uniform covering.

- Type of wrap if used:
None used

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. 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 material.

- Time after start of exposure:
15 Minutes post exposure

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
For the test material the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Classification of irritation potential is based upon relative tissue viability following the 15 minute exposure period followed by the 42 hour post-exposure incubation period according to the following:

Mean tissue viability is ≤50% : Irritant (I) R38

Mean tissue viability is >50% : Non-Irritant (NI)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15-minute exposure
Value:
36.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was 36.8% after a 15-minute exposure.
Other effects:
No

RESULTS

Direct MTT Reduction

The MTT solution containing the test material did not turn blue/purple which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.

The qualitative evaluation of tissue viability is given in Table 2.

Following the 15-minute exposure the test material treated tissues appeared blue/white which was considered indicative of viable tissue.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues and the standard deviation value of the percentage viability was ≤20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was ≥0.6 and the SD value of the percentage viability was ≤20%. The negative control acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

±SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

Negative Control Material

0.966

0.916

0.05

105.5

100*

0.912

99.6

0.870

95.0

Positive Control Material

0.085

0.073

0.01

9.3

8.0

0.078

8.5

0.057

6.2

Test Material

0.341

0.337

0.02

37.2

36.8

0.351

38.3

0.320

34.9


SD=    Standard Deviation

*=     The mean viability of the negative control tissues is set at 100%

 

Table2 : Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative Control Material

-

-

-

Positive Control Material

++

++

++

Test Material

+

+

+

MTT visual scoring scheme
-          =         blue tissue (viable)
+         =         blue/white tissue (semi-viable)
++       =         tissue is completely white (dead

Interpretation of results:
irritating
Remarks:
Migrated information EXAMPLE Criteria used for interpretation of results: expert judgment
Conclusions:
The test material was considered to be an Irritant.
Executive summary:
Introduction: The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTM reconstituted 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 reconstituted human epidermal cultures following topical exposure to the test material by means of the colourimetric 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 material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Methods:

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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-labelled 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 200µl samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm.

Data are presented in the form of percentage viability (MTT reduction in the test material treated tissues relative to negative control tissues).

Results: 

The relative mean viability of the test material treated tissues was 36.8% after a 15 -minute exposure.

Quality criteria: 

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

Conclusion:  The test material was considered to be an Irritant.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation
Remarks:
other: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed on 13 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
no guideline followed
Guideline:
other: The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential.
Deviations:
no
Principles of method if other than guideline:
The study was designed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye.
The rabbit enucleated eye test is used (in-house), as a first stage in the assessment of ocular irritancy potential. The preferred species of choice is the rabbit. The assay has undergone inter-laboratory validation and has been shown to reliably detect test materials that are negligible, or moderate to severe ocular irritants.
The strain of rabbit used in these laboratories has been shown to produce satisfactory responses using known ocular-irritants and non-ocular irritants during in-house validation (see attached Appendix 3). The results of the study are believed to be of value in predicting the ocular irritancy potential of the test material in man.
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1% w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.
Vehicle:
unchanged (no vehicle)
Remarks:
For the purpose of the study the test material was used as supplied.
Controls:
other: Two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).
Amount / concentration applied:
The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to the surface of the cornea.
Duration of treatment / exposure:
After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (approximately 32°C).
Observation period (in vivo):
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.

The thickness of the cornea was measured using an ultrasonic pachymeter. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.

The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment.

The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment.
Number of animals or in vitro replicates:
Three eyes were treated with test material, two additional eyes remained untreated for control purposes.
Details on study design:
Test Material Administration
Three eyes were treated with test material, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.
The test material was used undiluted as supplied. A volume of 0.1 ml of the test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 ml of saline solution (approximately 32°C).

Observations
Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment, according to the numerical evaluation given in Appendix 2 adopted from Advances in Modern Toxicology: Dermatoxicology, 4th Ed, (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815.
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation given in attached Appendix 2. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.
Irritation parameter:
cornea opacity score
Remarks:
Corneal Opacity
Basis:
other: Maximum score
Time point:
other: Maximum score
Max. score:
1
Reversibility:
not specified
Remarks:
N/A
Remarks on result:
other: Maximum score for Corneal Opacity 1
Irritation parameter:
cornea opacity score
Remarks:
Area of corneal opacity
Basis:
other: Maximum score
Time point:
other: Maximum score
Max. score:
3
Reversibility:
not specified
Remarks:
N/A
Remarks on result:
other: Maximum score for Area of corneal opacity 3
Irritation parameter:
cornea opacity score
Remarks:
Corneal Swelling (%)
Basis:
other: Mean score
Time point:
other: 60 Minutes
Score:
13.8
Reversibility:
not specified
Remarks:
N/A
Remarks on result:
other: Mean score @ 60 Minutes 13.8 %
Irritation parameter:
cornea opacity score
Remarks:
Corneal Swelling (%)
Basis:
other: Mean score
Time point:
other: 120 Minutes
Score:
35.3
Reversibility:
not specified
Remarks:
N/A
Remarks on result:
other: Mean score @ 120 Minutes 35.3 % which meets or exceeds cut-off value indicating a severe ocular irritant
Irritation parameter:
cornea opacity score
Remarks:
Corneal Swelling (%)
Basis:
other: Mean score
Time point:
other: 240 Minutes
Score:
58
Reversibility:
not specified
Remarks:
N/A
Remarks on result:
other: Mean score @ 240 Minutes 58.0 % which meets or exceeds cut-off value indicating a severe ocular irritant
Irritation parameter:
cornea opacity score
Remarks:
Condition of Corneal Epithelium
Basis:
other: Maximum score
Time point:
other: Maximum score
Reversibility:
not specified
Remarks on result:
other: Sloughing was seen which meets or exceeds cut-off value indicating a severe ocular irritant
Irritant / corrosive response data:
Corneal Opacity
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

Corneal Thickness and Condition
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3. The condition of the corneal epithelium following treatment is given in Table 4.
Corneal swelling of the test eyes during the study period was considerably greater than to that observed in the control eyes over the same period.
Sloughing of the corneal epithelium was noted in two test eyes. The condition of the corneal epithelium of one test eye and the control eyes appeared normal during the study period.

Fluorescein Uptake
Individual scores for fluorescein uptake are given in Table 5.
Fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*

REET Cut-Off Value

Maximum Corneal Opacity (Corneal Cloudiness x Area)

> or = 4

Maximum Fluorescein Uptake (Intensity x Area)

> or = 4

Mean Corneal Swelling (mins): 60, 120, 240

> or = 25%

Corneal Epithelium Observations

Any with pitting, mottling or sloughing

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

(mean corneal thickness post – treatment) – (mean corneal thickness post equilibration) x 100

Mean corneal thickness post equilibration

A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minutes post treatment observation periods.

A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.


*= Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant.

Table1              Individual Scores for Corneal Opacity

 

Test Eyes

Control Eyes

Chamber Number

1A

3A

5A

2A

4A

Time After Treatment (minutes)

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

60

120

180

240

Degree of Corneal Opacity

0

0

0

1

0

1

1

1

0

1

1

1

0

0

0

0

0

0

0

0

Area of Corneal Opacity

0

0

0

1

0

1

1

1

0

2

3

3

0

0

0

0

0

0

0

0

Table2              Individual Measurements for Corneal Thickness (µm)

Test Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

Chamber Number

1A

408

450

397

386

398

407.8

434

468

443

425

460

446.0

435

521

443

457

497

470.6

460

630

453

457

523

504.6

3A

426

423

401

420

422

418.4

441

587

425

551

529

506.6

455

657

431

592

612

549.4

463

677

444

682

693

581.0

5A

439

437

441

437

443

439.4

502

455

575

621

631

556.8

736

459

640

705

703

648.6

721

471

699

756

753

680.0

 

Control Eyes

Time After Treatment (minutes)

60

120

180

240

Corneal Position

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

oc

1

2

3

4

mean

Chamber Number

2A

389

353

362

383

362

369.8

386

337

365

373

359

364.0

369

343

345

360

352

353.8

363

329

343

355

354

348.8

4A

421

374

373

404

398

394.0

411

376

372

403

405

393.4

401

367

372

390

384

382.8

400

369

355

383

371

375.6

oc=    Optical centre

Table3              Determination of Corneal Swelling (%)

Test Eyes

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

1A

Post equilibration

347.8

N/A

3A

Post equilibration

368.0

N/A

5A

Post equilibration

397.4

N/A

60 Post treatment

407.8

17.3

60 Post treatment

418.4

13.7

60 Post treatment

439.4

10.6

120 Post treatment

446.0

28.2

120 Post treatment

506.6

37.7

120 Post treatment

556.8

40.1

180 Post treatment

470.6

35.3

180 Post treatment

549.4

49.3

180 Post treatment

648.6

63.2

240 Post treatment

504.6

45.1

240 Post treatment

581.0

57.9

240 Post treatment

680.0

71.1

 

Control Eyes

 

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Chamber Number

Observation Period (minutes)

Mean Corneal Thickness (µm)

Corneal Swelling (%)a

Test Eyes

Mean corneal swelling 1 hour following treatment 13.8%

Mean corneal swelling 2 hours following treatment 35.3%+

Mean corneal swelling 4 hours following treatment 58.0%+

2A

Post equilibration

353.2

N/A

4A

Post equilibration

384.4

N/A

60 Post treatment

369.8

4.7

60 Post treatment

394.0

2.5

Control Eyes

120 Post treatment

364.0

3.1

120 Post treatment

393.4

2.3

Mean corneal swelling 1 hour following treatment 3.6%

Mean corneal swelling 2 hours following treatment 3.8%

Mean corneal swelling 4 hours following treatment 0.0%

180 Post treatment

353.8

0.2

180 Post treatment

382.8

0.0

240 Post treatment

348.8

0.0

240 Post treatment

375.6

0.0

a=     % Corneal swelling =

N/A=  Not applicable

+ =    Meets or exceeds cut-off value and therefore indicates a severe eye irritant

Table4              Corneal Epithelium Condition

Test Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

1A

Normal

Normal

Normal

Normal

3A+

Normal

Sloughing

Sloughing

Sloughing

5A+

Normal

Sloughing

Sloughing

Sloughing

 

Control Eyes

Chamber Number

Time After Treatment (minutes)

60

120

180

240

2A

Normal

Normal

Normal

Normal

4A

Normal

Normal

Normal

Normal

+ = Meets or exceeds cut-off value indicating a severe ocular irritant

Table5              Individual Scores for Fluorescein Uptake (240 Minutes Post Dosing)

 

Test Eyes

Control Eyes

Chamber Number

1A

3A

5A

2A

4A

Intensity of Fluorescein Uptake

1

1

2

0

0

Area of Fluorescein Uptake

2

2

3

0

0

Interpretation of results:
highly irritating
Remarks:
Migrated information Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo. Criteria used for interpretation of results: expert judgment
Conclusions:
Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

Introduction. 

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

Methods. 

0.1 ml of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32°C ± 1.5°C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% Sodium Chloride).

Results.

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal Opacity

Fluorescein Uptake

Corneal Swelling (%)

Condition of Corneal Epithelium

Test Eyesa

Control Eyesb

Cldy

Area

Int

Area

60
mins

120
mins

240
mins

60
mins

120
mins

240
mins

1

3

2

3

13.8

35.3+

58.0+

3.6

2.7

0.0

Sloughing+

Conclusion. 

Following assessment of the data for all endpoints the test material was considered to have the potential to cause severe ocular irritancy in vivo.



a=      For each time point the swelling recorded is the mean of three eyes

b=      For each time point the swelling recorded is the mean of two eyes

Cldy=  Corneal cloudiness

Int=     Intensity of fluorescein uptake

Mins= Minutes following treatment

+=      Meets or exceeds cut-off value indicating a severe ocular irritant

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance was found to be non-corrosive to skin in the OECD 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis) Test. In the OECD 439 (In Vitro Skin Irritation: reconstructed Human Epidermis) Test skin irritation effects were noted for the test substance. The very slight erythema observed in rats up to 4-5 days in the acute dermal toxicity study indicates that there is potential for skin irritation and the material is classified and labelled accordingly (CLP Category 2).

 

The ocular irritation potential of the test substance was tested in a rabbit enucleated eye test (REET) in place of an in vivo eye irritation test because the test substance was suspected to be strongly irritating and/or corrosive. Treatment of enucleated rabbit eyes with the undiluted test substance for 10 seconds resulted in increased corneal swelling, damage to the corneal epithelium and corneal opacity. Assessment of the data for all endpoints indicated the notified chemical has the potential to cause severe ocular irritancy in vivo. Accordingly, it is classified and labelled as causing serious eye damage (CLP Category 1).


Justification for selection of skin irritation / corrosion endpoint:
Only one study available

Justification for selection of eye irritation endpoint:
Only one study available

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: highly irritating

Justification for classification or non-classification

The test data support classification of the registered substance as a skin irritant (Category 2) and severe eye irritant (Category 1) according to the criteria laid down in Regulation (EC) No 1272/2008 (i.e. CLP).

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