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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-19 to 2018-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation ornea-like Epithelium (RhCE) test method)
Version / remarks:
28 July 2015
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Version / remarks:
22 July 2015
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 irritation of chemicals
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: Reconstructed human cornea-like epithelium (RhCE) EpiOcular
Details on test animals or tissues and environmental conditions:
The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
1. Negative Control 50 μL Aqua dest.
2. Positive Control 50 μL methyl acetate
3. Test Item 50 μL (undiluted)
Duration of treatment / exposure:
30 +/- 2 min.
Duration of post- treatment incubation (in vitro):
- post soak incubation: 12 +/- 2 min.
- post treatment incubation: 120 +/- 15min
- MTT incubation: 3h
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per dose group.
Details on study design:
The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

Results and discussion

In vitro

Results
Irritation parameter:
other: Mean relative tissue viability [%]
Run / experiment:
Mean tissues 1 and 2
Value:
97.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
20.5
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
TEST ACCEPTANCE CRITERIA

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.814 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 20.5 < 50% pass
Max. Difference of % Viability [%] 1.4 < 20% pass

Any other information on results incl. tables

The mixture of 50 µl test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results

NSMTT [%] = [(ODKT- ODKU)/ODNK] * 100 = 0.99%

Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.

NSMTT1 [%] = [meanODKT1 - ODKU)/ODNK] * 100 = -0.34%

NSMTT2 [%] = [meanODKT2 - ODKU)/ODNK] * 100 = 2.32%

NSMTT1 - NSMTT2 =± 2.66%

NSMTT was ≤ 60% (0.99%) relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:

KCCV [%] = viabilityTM- NSMTT = 97.0%

 

The mixture of 50 µL test item per 1 mL A. dest showed colouring as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.

NSCliving [%] = [ODTVT/ODNK]*100 = 0.47%

Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.

NSC1 [%] = [ODTVT1/ODNK] * 100 = 0.39%

NSC2 [%] = [ODTVT2/ODNK] * 100 = 0.54%

NSC1 – NSC2 = ±0.15%

NSClivingwas ≤ 60% (0.47%) relative to the negative control of living epidermis and could therefore be used for determination of theNSC-corrected mean relative tissue viability (NSCCV) according to the following formula:

NSCCV [%] = viabilityTM[%] – NSCliving[%] = 96.4%

 

Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference.

The non-specificcolour of additional killed tissues (NSCkilled) was calculated according to the following formula:

NSCkilled [%] = [ODTKT/ODNK]*100 = 1.45%

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSClivingplus NSCkilled.

True Tissue Viability = [%] mean tissue viability – NSMTT –NSCliving+ NSCkilled= 97.9%

Result of the Test Item Sa 190

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570values

1.798

1.775

0.400

0.405

1.763

1.755

1.847

1.837

0.401

0.424

1.817

1.777

OD570values
(blank-corrected)

1.754

1.731

0.356

0.361

1.719

1.711

1.803

1.794

0.357

0.380

1.774

1.733

mean of the duplicates

1.778

1.762

0.356

0.371

1.746

1.722

mean OD

1.770*

0.364

1.734

TODTT- NSMTT

 -

 -

1.717

TODTTNSMTT und NSCliving

 -

 -

1.707

mean sd OD

0.011

0.010

0.017

tissue viability [%]

100.5

99.5

20.1

20.9

98.6

97.3

relative tissue viability difference [%]***

0.9

0.8

1.4

mean tissue viability [%]

100.0

20.5**

98.0

mean tissue viability [%]
- NSMTT corrected

 -

 -

97.0

mean tissue viability [%]
- NSMTT and NSClivingcorrected

 -

 -

96.4

True Tissue Viability

 -

 -

97.9

 

*              Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

**             Mean relative tissue viability of the positive control is < 50%

***            Relative tissue viability difference of replicate tissues is < 20%

Result of the NSMTT Control

NSMTT

KU

KT

Negative Control

Tissue

1

2

1

2

1

2

absolute OD570 -values

0.074

0.075

0.067

0.115

1.798

1.775

0.075

0.076

0.071

0.117

1.847

1.837

OD570(Blank Corrected)

0.030

0.031

0.023

0.071

1.754

1.731

0.031

0.032

0.027

0.073

1.803

1.794

mean OD570
(mean of 2 aliquots)

0.031

0.031

0.025

0.072

1.778

1.762

total mean OD570
(mean of the replicate tissues)

0.031

0.049

1.770

SD OD570(of the replicate tissues)

0.000

0.033

0.011

NSMTT [%]

0.99

 -

Relative Tissue Viability [%]

 -

100.5

99.5

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

0.6

CV [% Viabilities]

 -

0.6

Result of the NSCliving Control

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

absolute OD570 -values

0.050

0.053

2.098

2.048

0.051

0.055

2.111

2.021

absolute OD570 -
Blank corrected values

0.007

0.010

2.055

2.005

0.008

0.012

2.068

1.978

mean OD570
(mean of 2 aliquots)

0.008

0.011

2.062

1.991

total mean OD570
(mean of replicate tissues)

0.010

2.027

SD OD570
(of the 2 replicate tissues)

0.002

0.050

NSCliving[%]

0.47

 -

Relative Tissue Viability [%]

 -

101.7

98.3

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]***

 -

2.5

CV [% Viabilities]

 -

2.5

 

Result of the NSCkilled Control 

NSCkilled

TKT

Negative Control

Tissue

1

2

1

2

Absolute OD570 -values

0.065

0.078

2.098

2.048

0.064

0.082

2.111

2.021

Absolute OD570 -
Blank corrected values

0.022

0.035

2.055

2.005

0.021

0.039

2.068

1.978

Mean OD570
(mean of 3 aliquots)

0.022

0.037

2.062

1.991

Total Mean OD570
(Mean of Replicate Tissues)

0.029

2.027

SD OD570
(of the 2 replicate tissues)

0.011

0.050

NSCkilled[%]

1.45

 -

Relative Tissue Viability [%]

 -

101.7

98.3

Mean Relative Tissue Viability [%]

 -

100.0

SD Tissue Viability [%]

 -

2.5

CV [% Viabilities]

 -

2.5

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”
Executive summary:

In the present study the eye irritating potential of Sa 190 was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (97.9%; NSMTT-,NSCliving and NSCkilled-corrected).

The controls confirmed the validity of the study. The mean absolute OD570of the two negative control tissues was > 0.8 and < 2.5 (1.814). The mean relative tissue viability (% negative control) of the positive control was < 50% (20.5%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (1.4%).