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Description of key information

In a GLP compliant OECD TG 431 study, the test item was investigated for skin corrosive properties using the three-dimensional human skin model EpiDermTM, comprising a reconstructed epidermis with a functional stratum corneum. Based on the results, the test itemis considered to be corrosive to skin. The eye irritancy potential of the undiluted test substance was investigated in the bovine corneal opacity and permeability assay according to OECD TG 437 in consideration of GLP criteria. With regard to the irritation scores obtained in this in vitro assay, the test item has to be considered severely irritating to eyes. 

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:
experimental study
Adequacy of study:
key study
Study period:
2011-06-29 to 2011-07-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EpiDerm human epidermis model
Strain:
other: not applicable
Details on test animals and environmental conditions:
organotypic reconstituted three-dimensional model of the human epidermis
Type of coverage:
other: direct application
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
other: two tissues per treatment period for negative control (A. dest.) and positive control (8 N KOH)
Amount / concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 60 minutes
Number of animals:
two replicate tissues per treatment period
Details on study design:
2 replicate tissues per treatment period are dosed with the neat test item, the negative control (A. dest.) and the positive control (8 N KOH), repectively. After each treatment period the test item and controls are rinsed off and the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
< 50
Remarks on result:
other:
Remarks:
Basis: other: mean tissue viability of the negative control tissues. Time point: 3 min. Remarks: corrosive. (migrated information)
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
< 15
Remarks on result:
other:
Remarks:
Basis: other: mean tissue viability of the negative control tissues. Time point: 60 min. Remarks: corrosive. (migrated information)
Irritant / corrosive response data:
if mean tissue viability after 3 min is >= 50% and after 60 min is >= 15% of the respective negative control, the test item is classified as non-corrosive
Other effects:
The mixture of 100 µl test item per 1 ml MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple. Treatment of killed tissues with the test item showed that the test item was washed away before the addition of the MTT solution. Therefore, an influence on the assay was not detectable and a quantitative correction of results was not necessary.

3 min.

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

absolute OD550 -values

1.823

1.801

1.732

1.702

0.216

0.223

1.880

1.869

1.801

1.742

0.219

0.216

1.839

1.800

1.782

1.739

0.219

0.224

OD550(mean of 3 aliquots)

1.847

1.823

1.772

1.728

0.218

0.221

sd

0.029

0.040

0.036

0.022

0.002

0.004

OD550(mean of 2 replicate tissues)

1.835*

1.750

0.220

mean relative tissue viability [%]

100

95

12**

mean inter tissue viability difference [%]***

1.3

2.5

1.3

*       mean OD550 >= 0.8

**      mean relative tissue viability of the 3 min. positive control <= 30%

***     inter tissue viability difference <= 30%

60 min.

Name

Negative Control

Test Item

Positive Control

Tissue

1

2

1

2

1

2

absolute OD550 -values

1.915

1.898

0.265

0.243

0.181

0.157

1.920

1.927

0.271

0.246

0.182

0.160

1.956

1.824

0.263

0.245

0.182

0.153

OD550(mean of 3 aliquots)

1.930

1.883

0.266

0.245

0.182

0.157

sd

0.022

0.053

0.004

0.002

0.001

0.003

OD550(mean of 2 replicate tissues)

1.907*

0.255

0.169

mean relative tissue viability [%]

100

13

9

mean inter tissue viability difference [%]***

2.5

8.5

14.8

*       mean OD550 >= 0.8

***     inter tissue viability difference <= 30%

Interpretation of results:
corrosive
Remarks:
Migrated information Criteria used for interpretation of results: other: OECD 431
Conclusions:
The test item is classified as "corrosive".
Executive summary:

The potential of the test item to induce skin corrosion was analysed by using the three-dimensional human skin model EpiDermTM, comprising a reconstructed epidermis with a functional stratum corneum. The undiluted substance was applied topically to the EpiDermTM tissue for 3 minutes and 60 minutes respectively, followed by immediate determination of cytotoxic effects via the MTT reduction assay. Corrosivity potential was predicted from the relative mean tissue viabilities obtained after both treatment periods compared to the corresponding negative control tissues concurrently treated with A. dest (distilled water).

The test item showed corrosive effects in that the mean relative tissue viability (% negative control) was > 50% (95%) after3 minute treatment and < 15% (13%) after 60 minute treatment. The controls confirmed the validity of the study. The mean OD550 of the two negative control tissues was > 0.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 30% (12%) after 3 minute treatment. The maximum inter tissue difference of replicate tissues of all dose groups was < 30% (1.3% - 14.8%).

Based on the results of this study, the test item showed corrosive effects. The test item therefore is classified as "corrosive".

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-07-20 to 2012-01-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: OECD Guidline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants), adopted: 7 September 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: not applicable
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
not applicable
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
750 microL of the undiluted test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 10 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed.
Details on study design:
Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals
(from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep
on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera.
The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France)
with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective
cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws.
The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed
into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1
was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the
measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer
was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated.
The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity
measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity
readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of
each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed.
The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium
washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed
with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with
fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were
incubated for 90 minutes at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density
at 490 nm (OD490) was determined, using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard
deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading.
These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas.
The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500),
were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article
and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected
OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for
that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
140.44
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The in vitro score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Opazität
Cornea-No. Test Item Opacity Opacity Change of Corrected
    blank value post dose opacity values opacity values
1 Negative 3 4 1  
2 Control 3 2 -1  
3   3 4 1  
MV   3.00 3.33 0.33  
4 Positive 3 80 77 76.67
5 Control 3 67 64 63.67
6   3 65 62 61.67
MV   3.00 70.67 67.67 67.33
7 Test item 2 110 108 107.67
8 Genamin 2 111 109 108.67
9 CH 020 2 113 111 110.67
MV   2.00 111.33 109.33 109.00

Permaebilität
Cornea-No. Test Item OD490 Corrected
      OD490 values
1 Negative 0.008  
2 Control 0.005  
3   0.005  
MV   0.006  
4 Positive 1.016 1.010
5 Control 0.967 0.961
6   1.432 1.426
MV   1.138 1.132
7 Test item 2.111 2.105
8 FHP-OHS 2.088 2.082
9   2.107 2.101
MV   2.102 2.096

in vitroScore
Cornea-No. Test Item Corrected Corrected  in vitro
    opacity value OD490 value Score
1 Negative 1 0.008  
2 Control -1 0.005  
3   1 0.005  
MV   0.33 0.006 0.42
4 Positive 76.67 1.010  
5 Control 63.67 0.961  
6   61.67 1.426  
MV   67.33 1.132 84.32
7 Test item 107.67 2.105  
8 FHP-OHS 108.67 2.082  
9   110.67 2.101  
MV   109.00 2.096 140.44
Interpretation of results:
highly irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the evaluation criteria the test item is classified as very severe eye irritant.
Executive summary:

The eye irritancy potential of the undiluted test substance was investigated in the bovine corneal opacity and permeability assay and the mean in vitro score was calculated to be 140.44. Based on the results of this study a mean in vitro score of 140.44 was obtained. The test substance therefore is classified as severe eye irritant. The in vitro score obtained with the positive control was within the current historical mean and therefore this assay is considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item was tested for skin corrosion in the in vitro human skin model test according OECD TG 431and in the in vitro membrane barrier test according to OECD TG 435. Both test systems revealed that the test item should be regarded as corrosive to skin. With respect to potential eye irritation, the test item was evaluated in the bovine corneal opacity and permeability assay (BCOP) following OECD test guideline 437. Based on the scores revealed in this in vitro assay, it is concluded that the test item is severely irritating to eyes. The above findings from the in vitro test systems and the concluded severe irritative / corrosive properties of the test item are most probably attributable to the basicity of the submission substance.


Justification for selection of skin irritation / corrosion endpoint:
Guideline study according to GLP with a Klimisch rating 1.

Justification for selection of eye irritation endpoint:
Guideline study according to GLP with a Klimisch rating 1.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: highly irritating

Justification for classification or non-classification

In two guideline-compliant in vitro studies according to OECD TG 431 and OECD TG 435, the test item showed corrosive effects. The test substance is classified as “corrosive”, subcategory 1C according to GHS-CLP (equivalent to EU Risk-Phrase R34 according to DSD), as the mean time to activate the chemical detection system which was covered by a bio-barrier membane was > 60 - 240 min (category 1).

With regard to eye irritation, a bovine corneal opacity and permeability test for the identification ocular corrosives and severe irritants revealed irritation scores triggering a classification as causing serious damage to eyes (category 1) according to GHS-CLP (equivalent to EU Risk-Phrase R41 according to DSD).

Although no conclusion on classification and labelling regarding respiratory tract irritation can be derived due to lack of data, classification of the submission substance as respiratory irritant (category 3) according to GHS-CLP (equivalent to EU Risk Phrase R37) seems to be appropriate when taking all data on the irritative properties of the test item into account.