Histidine

Eye irritation

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 guideline

Test guideline 1

Qualifier:

according to guideline

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

Test guideline 2

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Histidine

EC Number:

200-745-3

EC Name:

Histidine

Cas Number:

71-00-1

Molecular formula:

C6H9N3O2

IUPAC Name:

histidine

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

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.