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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:
17-11-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 2010
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Chemical name: L-Histidine
- Molecular formula: C6H9N3O2
- Molecular weight: 155.15 g/mol
- Appearance: White crystalline powder
- Purity: NMT 0.2% impurities (HPLC)
- Homogeneity: homogeneous
- Stability: H2O: unknown; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
- Solubility: H2O: >1g/L; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
- Batch: P816P001
- Storage: Room Temperature (20 ± 5 °C); Keep away from light; Keep away from humidity
- Expiry date: 2019-01

Test animals / tissue source

Species:
cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany.
Bovine eyes were obtained on the day of the test.
Age of cattle: 12-60 months.
Transport to test facility in Hank's balanced salts solution supplemented with 0.01% streptomycin and 0.01% penicillin.
After dissection the corneas are incubated in medium at 32 +/- 1°C for 1h.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Replicate 1: 506.8 mg
Replicate 2: 503.4 mg
Replicate 3: 508.2 mg
Duration of treatment / exposure:
4 hours exposure time at 32 +/-1°C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 +/- 1°C (for premeability measurement)
Number of animals or in vitro replicates:
3 test item replicates, 3 negative control replicates and 3 positive control replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Only corneas free from damage were used.
Corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside.
Each cornea was transferred to a cornea holder in which pre-warmed (32+/-1°C) cMEM without phenol was filled.
The holders were then incubated for 1 hour in the incubation chamber at 32+/-1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Quality check after initial incubation: the medium was changed and the baseline opacity for each cornea was recorded.
None of the cornea showed tissue damage, therefore all corneas were used.

TREATMENT METHOD:
Open chamber method.
The solid was applied directly on the cornea, in such a manner that as much as possible of the cornea was covered with test item.

REMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATION AND MEASURING OPACITY AND PERMEABILITY
Thorough rinsins with cMEM with phenol red, followed by rinsing with cMEM without phenol red.
Subsequently, both chambers were filled with cMEM without phenol red and the final opacity value of each cornea was recorded.
The cMEM without phenol red was then removed from the front chamber and 1 ML sodium fluorescein solution (5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32+/-1°C.
After incubation, the content of the posterior chamber was thoroughly mixed. Then the permeability of the cornea was measured as optical density of the liquid at 492 nm.

SCORING SYSTEM:

1) Opacity = [(I0/I)-b]a

with:
a = 0.0251 (opacitometer-specific, empirically determined)
b = 0.9894 (opacitometer-specific, empirically determined)
I0 = empirically determined illuminance through a cornea holder with windows and medium (expressed in LUX) (here: I0 = 1078.3)
I = measured illuminance (expressed in LUX)

2) Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by substracting the mean negative control conrea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final OD492 values of the treated corneas for one treatment group.

3) In Vitro Irritancy Score (IVIS)

For the negative control:
IVIS = opacity difference + (15 x corrected OD492 value)

For the positice control and the test item treatment group:
IVIS = (opacity difference - mean opacity difference of the negative control) + (15 x (OD492 - mean OD492 of the negative control))

DECISION CRITERIA:

IVIS <= 3: no category
IVIS >3 and <= 55: no prediction can be made
IVIS > 55: eye damage Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3 replicates
Value:
0.32
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 1
Value:
-0.46
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 2
Value:
0.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 3
Value:
1.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Criterion: IVIS <= 3
- Found: IVIS = -0.04

- Acceptance criteria met for positive control: yes
- Criterion: IVIS = 73.16-164.28
- Found: IVIS = 118.15

Any other information on results incl. tables

Opacity Values:

 

Treatment group

Replicate

Before exposure

After exposure

Negative control

1

1026

1019

2

986

1030

3

1009

1016

Test item

1

1021

1037

2

1026

1021

3

1031

2008

Positive control

1

1008

334

2

995

441

3

997

350

 

Optical density at 492 nm of Blank:

 

Measurement

cMEM without phenol red

1

0.033

2

0.031

3

0.039

Mean

0.034

 

Permeability Values:

 

Repl.

Measurem. #

Measured value

Measured value- blank

Mean of replicate

Mean of 3 replicates

Corrected

Neg. contr.

1

1

2

3

0.046

0.042

0.046

0.0117

0.0077

0.0117

0.0103

0.0388

--

2

1

2

3

0.044

0.042

0.042

0.0097

0.0077

0.0077

0.0083

--

3

1

2

3

0.137

0.134

0.125

0.1027

0.0997

0.0907

0.0977

--

Test item

1

1

2

3

0.043

0.045

0.045

0.0087

0.0107

0.0107

0.0100

--

-0.0288

2

1

2

3

0.035

0.035

0.037

0.0007

0.0007

0.0027

0.0013

-0.0374

3

1

2

3

0.042

0.042

0.050

0.0077

0.0077

0.0157

0.0103

-0.0284

Pos. contr.

1

1

2

3

0.724

0.720

0.714

0.6897

00.6857

0.6797

0.6850

--

3.3862

2

1

2

3

0.576

0.576

0.576

0.5417

0.5417

0.5417

0.5417

2.6696

3

1

2

3

0.605

0.601

0.594

0.5707

0.5667

0.5597

0.5657

2.7896

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the outcome of the BCOP assessment, L-histidine was found to be not irritant or corrosive to the eyes.
Executive summary:

The potential of L-HISTIDINE to cause eye irritation or eye dammage was assessed with the in vitro BCOP assay. The study was performed according to OECD 437 and GLP.

The positive and negative control results met the criteria, indicating that the test system was valid.

The IVIS score for the test item was calculated to be 0.32. As this is below the threshold value of 3, L-histidine was found to not induce effects to the eye.