Ammonium glycyrrhizate

Endpoint summary

Key value for chemical safety assessment

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

The 0.60 cm2 Reconstructed human Cornea-like Epithelia were received on 17 october 2017. The same day, the tissues in their well shipping container were equilibrated at room temperature for 15 minutes. Then, the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filyer. They were places in 6 wells culture plate with which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 19 hours and 20 minutes at standard culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours and 15 minutes

Number of animals or in vitro replicates:

2 replicates (RhCE)

Details on study design:

The test item was applied on a nylon mesh at an approximate dose of 50mg to the entire surface of 2 living RhCE tissues replicate during 6 hours at standard culture conditions.

In the same experimental conditions, a positive control (Methyl acetate) and a negative control (distilled water) were carried out. The controls were applied, as supplied, at the dose of 50µL, to the surface of 2 RhCE tissue replicates during 6 hours at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+ Mg2+ Free-DPBS. The rinsed tissues were checked for any coloration and noted to be of comparable colour with the negative control treated tissues (whitish). The rinsing step was followed by a 25-minutes-post-exposure immersion period at room temperature in 5mL of fresh medium to remove any test item absorbed into the tissue.

The RhCE constructs were then incubated for an 18 hours and 15 minutes post-exposure incubation at standard culture conditions in 1mL of fresh medium at 37 °C, 5% CO2.

Viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects ad appearance of clear cytotoxic effects. The RhCE tissue viability was measured by enzymatic covnersion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues. The RhCE constructs were places in 300µL of a MTT solution at 1.0 mg/ml for 3 hours and 55 minutes at standard culture conditions. The precipitated blue formazan products was then extracted from the tissues by placing each insert in 2mL of isopropanol during 2 hours and 08 minutes at room temperature in the dark.
The concentration of formazan was measured by determining the optical density at 570 nm, just after dilution of the extraction in isopropanol (1:2)
The optical density was measured in triplicate samples of formazan extracts. The measured optical density are proportional to the number of living cells. The measurment was performed using the ELx800 absorbance microplate reader.

Irritation parameter:

other: Viability %

Value:

12.32

Negative controls validity:

valid

Remarks:

100.00%

Positive controls validity:

valid

Remarks:

19.83%

Interpretation of results:

other: Based on a part of integrated approach, these results support a classification in Category 2 or Category 1 of GHS.

Conclusions:

Based on OECD 492 (2015), GLP study, results are considered scientifically valid to be used as part of an integrated approach to support a classification in Category 2 or Category 1 according to GHS and CLP regulation.

Refer to endpoint summary for final conclusion.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
The eyes collected from chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).

Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 28 August 2017 at 8:15 am. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
The eyes were enucleated on 28 August 2017 at 9:35 am.

The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the
nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.9°C.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL

POSTIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 mg

Duration of treatment / exposure:

Test item
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied,as supplied, to the cornea such that the entire surface of the cornea was evenly covered with the test item.

After 10 seconds, the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, an additional rinse was performed with 2x10 mL of physiological saline. A cotton swab moistened with physiological saline was used in order to gently remove the test item from the cornea but residue of the test item remained on the surface of the cornea, which did not prevent observations.

The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

Controls
Concurrent negative control (physiological saline) and positive control (sodium hydroxide) were included in this experiment. One eye was treated with the negative control and three eyes were treated with the positive control.

Number of animals or in vitro replicates:

3 eyes treated with test item.
1 eye treated for negative control
3 eyes treated for positive control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

EQUILIBRATION AND BASELINE RECORDINGS
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.9°C.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these
criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3 eyes tested with test item.

NEGATIVE CONTROL USED
Concurrent, physiological saline

POSITIVE CONTROL USED
Concurrent, sodium hydroxide

APPLICATION DOSE AND EXPOSURE TIME

OBSERVATION PERIOD
All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm. Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
After treatment, eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, an additional rinse was performed with 2x10 mL of physiological saline. A cotton swab moistened with physiological saline was used in order to gently remove the test item from the cornea but residue of the test item remained on the surface of the cornea, which did not prevent observations.
The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.


METHODS FOR MEASURED ENDPOINTS:
- Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements according to the following from TG. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall
category score was then given for each test item.

- Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item. The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM: according to TG OECD 438 (2013).

DECISION CRITERIA: according to TG OECD 438 (2013) .

Irritation parameter:

cornea opacity score

Remarks:

max score

Value:

0.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result: ICE class II

Irritation parameter:

fluorescein retention score

Remarks:

mean score at 30 min

Value:

2.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result: ICE class IV

Irritation parameter:

percent corneal swelling

Remarks:

max score

Value:

7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result ICE: class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no morphological effects were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on OECD 438 (2013), GLP study, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by OECD guideline (2013).

Refer to endpoint summary for final conclusion.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Two studies on endpoint eye irritation have been provided in IUCLID dossier:

- OECD 438, GLP study, Klimisch 1 whith the conclusion :"no prediction can be made";

- OECD 492, GLP study, Klimisch 1 with the conclusion : "a classification in Category 2 or Category 1 according to GHS and CLP regulation is supported".

Otherwise , according to the Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a : Endpoint specific guidance (Version 6.0, July 2017), in case, inconsistent in vitro results for serious eye damage/ eye irritation are obtained, the Weight of Evidence including skin irritation (Category 2) may support the classification for eye irritation (Category 2), as a precautionary principle (Section R.7.2.11.2. Testing and assessment strategy for serious eye damage/ eye irritation.

A skin irritation study (OECD 439, GLP study, klimisch 1) has been provided in IUCLID dossier and indicated a non-classification for this endpoint. Therefore, according to paragraph above, a classification for eye irritation, Category 2; H319 in GHS and CLP Regulation is warranted for a conservative approach.