5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-benzopyrone

Endpoint summary

Description of key information

Skin irritation. Key study. Method according to OECD 439, GLP study. The mean corrected percent viability of the SkinEthic™ RHE treated tissues was 69.7%, versus 1.0% in the positive control. Therefore, the test item is not irritant to the skin.

Eye irritation. Key study. Method according to OECD 438, GLP study. According to the overall in vitro classification, the test item does not require classification for eye irritation, since the combinations of the 3 endpoints for the test item were 3xI (No category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 16th to December 1st, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: normal human keratinocytes.

Cell source:

other: human adult donor, not specified.

Source strain:

not specified

Details on animal used as source of test system:

The SkinEthic™ RHE model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 16-RHE-124
- Delivery date: 29/11/2016
- Expiration date: 05/12/2016
- Date of initiation of testing: 16/11/2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.3 (CV 1.3%)
- Barrier function: 4.3 h
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no interference.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 12 minutes.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

69.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(DPBS)

Positive controls validity:

valid

Remarks:

1.0% viability (5% SDS)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The standard deviation of the negative control group was 21.7%, instead of ≤ 18% as initially scheduled. However, considering the results obtained, this deviation is considered as without impact on the conclusion of the study.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1. Summary of results.

	Skin	OD	Mean OD/ disc (#)	Mean OD/ product	Viability %	Mean viability %	SD	Conclusion
Negative control	1	1.541 1.650 1.668	1.619	1.316	123.1	100.0	21.7	-
	2	1.268 1.271 1.290	1.276		97.0
	3	1.032 1.044 1.082	1.052		80.0
Positive control	4	0.013 0.016 0.013	0.014	0.013	1.1	1.0	0.1	Irritant
	5	0.015 0.013 0.013	0.013		1.0
	6	0.012 0.011 0.011	0.011		0.8
Test ítem PH-16/0575	24	0.895 0.957 0.966	0.939	0.918	71.4	69.7	1.6	Non irritant
	25	0.898 0.899 0.893	0.896		68.1
	26	0.912 0.903 0.940	0.918		69.8

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corrected percent viability of the treated tissues was 69.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 41 hours 12 minutes in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under test conditions, the mean corrected percent viability of the treated tissues was 69.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 21st, 2016.

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

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.
- Time interval prior to initiating testing: The heads were collected on 21 November 2016 at 8:15 am. The eyes were enucleated at Phycher on 21 November 2016 at 9:40 am.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

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

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3 replicates per group (test item, positive control, negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage. 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.

EQUILIBRATION AND BASELINE RECORDINGS: Once all eyes had been examined and approved, the eyes were incubated between 45 and 65 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

NEGATIVE CONTROL USED: 30 µL physiological saline – Dutscher Batch No. 3012316.

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED: 30 mg sodium hydroxide – Sigma, Batch No. MKBP7805V.

APPLICATION DOSE AND EXPOSURE TIME: 30 mg test item, exposure 10 seconds.

OBSERVATION PERIOD: 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: the test item was rinsed from the eye with 20 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, 2 additional rinses were performed with 10 mL of physiological saline. A cotton swab moistened with physiological saline was used to gently remove all the test item from the cornea. A last rinse was performed with 10 mL of physiological saline.
- Indicate any deviation from test procedure in the Guideline: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Damage to epithelium based on fluorescein retention: 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.
- Swelling: measured with optical pachymeter on a slit-lamp microscope (HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I); slit-width setting: the slit-width was set at 9½, equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. The value was determined from corneal thickness measurements. 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.
- Macroscopic morphological damage to the surface: qualitative assessment. The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.
- Others (e.g, histopathology): no.

SCORING SYSTEM:
- Mean corneal swelling (%) was calculated as follows: [(corneal thickness at time t - corneal thickness at time 0)/corneal thickness at time 0] x 100.
- Mean maximum opacity score:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris clearly visible
2 Easily discernible translucent area; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment:
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: as indicated in the TG.

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

cornea opacity score

Run / experiment:

max mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

percent corneal swelling

Run / experiment:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class I

Irritation parameter:

morphological effects

Run / experiment:

mean

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:

OTHER EFFECTS:
- Visible damage on test system: no. No morphological effects were noted, whatever the examination time. Rest of product not preventing the evaluation of corneal opacity, fluorescein retention and corneal thickness noted at 30 min post-dose in eyes No. 4, 5, 6.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

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.

Table 1. Test item results (30 mg), 21/11/2016.

Endpoint measured	Eye No.	0	30	75	120	180	240
Corneal opacity	4	0	0	0	0	0	0
	5	0	0	0	0	0	0
	6	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class		I
Fluorescein retention	4	0.5	0.5	-	-	-	-
	5	0.5	0.5	-	-	-	-
	6	0.5	0.5	-	-	-	-
Mean		0.5	0.5	-	-	-	-
ICE class		I
Corneal thickness	4	0.60	0.60	0.60	0.60	0.60	0.60
	5	0.58	0.58	0.58	0.58	0.58	0.58
	6	0.59	0.59	0.59	0.59	0.59	0.59
Corneal swelling (%)	4	-	0	0	0	0	0
	5	-	0	0	0	0	0
	6	-	0	0	0	0	0
Mean		-	0	0	0	0	0
ICE class		I
Combination of the 3 endpoints		3 x I
Classification		No category

No morphological effects were noted, whatever the examination time.

Rest of product not preventing the evaluation of corneal opacity, fluorescein retention and corneal thickness noted at 30 min post-dose in eyes No. 4, 5, 6.

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The combinations of the 3 endpoints for the test item were 3 x I. Therefore, the test item does not cause eye damange.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30 µL of physiological saline (negative control). Three eyeballs were used in each group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to the overall in vitro classification (UN GHS), the test item does not require classification for eye irritation, since the combinations of the 3 endpoints for the test item were 3xI (No category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. After 42h post-incubation, the viability of each tissue was assessed by incubating the solution with MTT, extracting the precipitated formazan crystals, and determing the OD spectrophotometrically. Under test conditions, the mean corrected percent viability of the treated tissues was 69.7%, versus 1.0% in the positive control. Therefore, the test item is not irritant to the skin.

Eye irritation. Key study. An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 (GLP study). Three eyeballs per group were isolated from chickens and, after appropriate preparation, were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30µL of physiological saline (negative control). According to the overall in vitro classification, the test item does not require classification for eye irritation, since the combinations of the 3 endpoints for the test item were 3xI (No category).

Justification for classification or non-classification

Skin irritation: Based on the available information (69.7 % tissue viability), the test item is not classified for skin irritation/corrosion, in accordance with CLP Regulation (EU) No. 1272/2008.

Eye irritation: Based on the available information (No category based on 3 x I), the substance does not cause serious eye damage (Category 1) according to CLP Regulation (EC) No. 1272/2008.