Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-06-28 to 2019-07-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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 or serious eye damage.
Version / remarks:
18 June 2019
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
Version / remarks:
22 July 2015
Qualifier:
according to
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
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
Name: cyclic Glucamide C8-C10
Chemical Name: Amides, C8-C10, N-(1-deoxy-D-glucitol-1-yl)-N-methyl, dehydrated (CAS name)
Batch No.: BP 1c (reactor mix)
CAS No 2290526-77-9
Compounds: UVCB
main compounds: C15H29NO5 (C8-acyl-derivative)
C17H33NO5 (C10-acyl-derivative)
8.5% unidentified components
Aggregate State at RT: highly viscous mass (20°C)
Colour: brown
Storage Conditions: room temperature, dry
Expiry Date: 28 March 2021
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test system

Amount / concentration applied:
50 µL
Duration of treatment / exposure:
incubation: 30 +/- 2 min.
Observation period (in vivo):
post soak: 12 +/- 2 min.
post treatment: 120 +/- 15 min.
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:
1
Value:
4.2
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:
Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.924 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 47.9 < 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. The killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT- ODKU)/ODNC] * 100 = [(0.042-0.093)/1.877] = -2.72%

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

NSMTT1 [%] = [meanODKT1- ODKU)/ODNC] * 100 = [(0.042-0.093)/1.877] = -2.72%

NSMTT2 [%] = [meanODKT2- ODKU)/ODNC] * 100 = [(0.042-0.093)/1.877] = -2.72%

NSMTT1 - NSMTT2 = 0%

NSMTT was ≤ 60% (-2.72% NSMTT) 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 = 1.6% - (-2.72%) ≈ 4.2%

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent.Therefore, NSCliving equalled 0%.

Result of the Test Item cyclic Glucamide C8-C10

Name

Negative Control

Positive Control

Test Item

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

1.921

1.887

0.939

0.944

0.074

0.075

1.953

1.933

0.940

0.956

0.077

0.076

Mean Absolute OD570

1.924****

0.945

0.076

OD570(Blank Corrected)

1.874

1.840

0.893

0.898

0.028

0.029

1.907

1.887

0.894

0.910

0.031

0.029

Mean OD570of the Duplicates
(Blank Corrected)

1.891

1.864

0.893

0.904

0.029

0.029

Total Mean OD570of the 2 Replicate Tissues (Blank Corrected)

1.877*

0.899

0.029

TODTT

 -

 -

0.079

SD of Mean OD570of the Duplicates (Blank Corrected)

0.019

0.008

0.000

Relative Tissue Viability [%]

100.7

99.3

47.6

48.2

1.6

1.5

Relative Tissue Viability
Difference [%]***

1.4

0.6

0.0

Mean Relative Tissue Viability [%]

100.0

47.9**

1.6

Mean Relative Tissue Viability [%]
- NSMTT corrected

 -

 -

4.2

*              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%

****        Mean absolute OD570 of the negative control is ≥ 0.8 and ≤ 2.5     

  Result of the NSMTT control 

NSMTT

KU

KT

Negative Control

Replicate Tissue

1

2

1

2

1

2

Absolute OD570

0.146

0.128

0.086

0.087

1.921

1.887

0.151

0.131

0.091

0.090

1.953

1.933

OD570(Blank Corrected)

0.100

0.081

0.040

0.041

1.874

1.840

0.104

0.085

0.045

0.044

1.907

1.887

Mean OD570of the Duplicates
(Blank Corrected)

0.102

0.083

0.042

0.042

1.891

1.864

Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected)

0.093

0.042

1.877

SD of Mean OD570of the Duplicates (Blank Corrected)

0.014

0.000

0.019

NSMTT [%]

-2.7

 -

Relative Tissue Viability [%]

 -

100.7

99.3

Relative Tissue Viability
Difference [%]

 -

1.4

Mean Relative Tissue Viability [%]

 -

100.0

Applicant's summary and conclusion

Conclusions:
In this study under the given conditions the test item showed irritant effects.
Executive summary:

In the present study cyclic Glucamide C8-C10 was applied topically to the EpiOcular TM tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min post-treatment period cytotoxic effects were determined via MTT reduction assay.

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

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. The killed tissue controls were performed for quantitative correction of results.

NSMTT [%] = [(ODKT- ODKU)/ODNC] * 100 = [(0.042-0.093)/1.877] = -2.72%

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

NSMTT1 [%] = [meanODKT1- ODKU)/ODNC] * 100 =[(0.042-0.093)/1.877] = -2.72%

NSMTT2 [%] = [meanODKT2- ODKU)/ODNC] * 100 =[(0.042-0.093)/1.877] = -2.72%

NSMTT1 - NSMTT2 =0%

NSMTT was ≤ 60% (-2.72% NSMTT) 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 = 1.6% - (-2.72%) ≈ 4.2%

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (4.2% NSMTT-corrected).

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.924). The mean relative tissue viability (% negative control) of the positive control was < 50% (47.9%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (1.4%).