Hesperidin

Endpoint summary

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study without deviations

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: seeded human epidermal keratinocytes

Strain:

other: supplied by SkinEthic Laboratories, Nice, France

Details on test animals or test system and environmental conditions:

The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Type of coverage:

other: EPISKIN model

Preparation of test site:

not specified

Controls:

other: sodium dodecyl sulphate (SDS) 5% as positive control

Amount / concentration applied:

Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Observation period:

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 ºC, 5% CO2 in air for 42 hours.
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was
transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination. 2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO 2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Number of animals:

not relevant, measurements were made in triplicates for negative control, positive control and test item.

Details on study design:

Absorbance/Optical Density measurement at day 6:
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

other: other: Relative mean viability

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: viability of negative control. Time point: 6 days following incubation. Max. score: 0.0. Reversibility: no data. Remarks: Max score is theoretical for no viability. (migrated information)

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.7%. The positive control acceptance criterion was therefore satisfied. The mean OD₅₄₀ for the negative control treated tissues was 0.841 and the standard deviation value of the percentage viability was 7.0%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 6.0%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item hesperidin was considered to be Non-Irritant (NI) in this human epidermis skin model test.

Executive summary:

The relative mean viability of the test item treated tissues was 100.0% after a 15-Minute exposure period. Thus, hesperidin had no effect on viability and the substance is considered non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study without significant deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.27 or 2.36 kg and were twelve to twenty weeks old. After an acclimatisation period of at least five days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A volume of 0.1 ml of the test item, which was found to weigh approximately 75 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.

Duration of treatment / exposure:

72 hours in total with score reading following 1, 24, 48 and 72 hours after initial exposure.

Observation period (in vivo):

Terminated after 72 hours due to lack of irriation.

Number of animals or in vitro replicates:

2 animals were used in this experiment

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A volume of 0.1 ml of the test item, which was found to weigh approximately 75 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.
After consideration of the ocular responses produced in the first treated animal, a second animal was treated. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (from Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded. Individual bodyweights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Due to the very weak response regards irritation, a third test animal was not required and the test was terminated as the substance was shown to be not an eye irritant.

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

mean

Remarks:

of 2 animals

Time point:

other: 1 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

mean

Remarks:

of 2 animals

Time point:

24 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

mean

Remarks:

of 2 animals

Time point:

48 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

mean

Remarks:

of 2 animals

Time point:

72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Remarks:

opacity

Basis:

mean

Remarks:

of 2 animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 2 animals

Time point:

other: 1 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 2 animals

Time point:

24 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 24 h

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 2 animals

Time point:

48 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 2 animals

Time point:

72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 2 animals

Time point:

24/48/72 h

Score:

0.33

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of 2 animals

Time point:

other: 1 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of 2 animals

Time point:

24 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of 2 animals

Time point:

48 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 h

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of 2 animals

Time point:

72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Remarks:

of 2 animals

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 2 animals

Time point:

other: 1 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 2 animals

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 2 animals

Time point:

48 h

Score:

0.5

Max. score:

4

Reversibility:

fully reversible within: 24 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 2 animals

Time point:

72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 2 animals

Time point:

24/48/72 h

Score:

0.5

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

As both rabbits tested showed corneal opacity <1, iritis <1, conjunctival redness < 2 and conjunctival oedema (chemosis) <2, calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and all scores on both animals returned to zero within 72 hours, the substance is not subject to classification according to GHS (Globally Harmonized System of Classification and Labelling of Chemicals) and CLP (Regulation (EC) No 1272/2008). As a result, for animal welfare, a third animal was not used in this test.

No corneal effects were noted during the study. Iridial inflammation was noted in both treated eyes one and 24 hours after treatment. Moderate conjunctival irritation was noted in both treated eyes one hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24-Hour observation with minimal conjunctival irritation noted in both treated eyes at the 48-Hour observation. Both treated eyes appeared normal at the 72-Hour observation.

Both animals showed expected gain in bodyweight during the study.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

As a result of this test hesperidin is not subject to classification for eye-irriation according to CLP and/or DSD .

Executive summary:

As both rabbits tested showed corneal opacity <1, iritis <1, conjunctival redness < 2 and conjunctival oedema (chemosis) <2, calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and all scores on both animals returned to zero within 72 hours, the substance is not subject to classification according to GHS (Globally Harmonized System of Classification and Labelling of Chemicals) and CLP (Regulation (EC) No 1272/2008). As a result, for animal welfare, a third animal was not used in this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation:

Hesperidin has been investigated in an EPISKIN OECD 439 respectively Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was proven to be non-irritant in this test. The EU Commission has acknowledged in its statement on the validity of in-vitro tests for skin irritation at its 26th meeting, held on 26-27th of April 2007 for the Validation of Alternative Methods (ECVAM) unanimously that the EPISKIN method showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction, and for being used as a replacement for the in-vivo Draize Skin Irritation Test . Consequently, for animal welfare an in-vivo test is not scientifically justifiable.

Eye corrosion/damage:

Hesperidin has been investigated in an integrated testing strategy for its effects on eyes.

First a BCOP test according to OECD 437 had been performed showing the substance not to be corrosive. In order to decide whether hesperidin may be irritant to eyes an in-vivo eye irritation study according to OECD 405 was performed. As both rabbits tested in this study showed corneal opacity <1, iritis <1, conjunctival redness < 2 and conjunctival oedema (chemosis) <2, calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and all scores on both animals returned to zero within 72 hours, the substance is not subject to classification according to GHS (Globally Harmonized System of Classification and Labelling of Chemicals) and CLP (Regulation (EC) No 1272/2008). As a result, for animal welfare, a third animal was not used in this test.

Justification for selection of skin irritation / corrosion endpoint:
fully compliant in-vitro skin irritation test, performed according to GLP and adequate for predicting skin irritation properties.

Justification for selection of eye irritation endpoint:
fully compliant in-vivo eye irritation study performed according to GLP

Justification for classification or non-classification

Hesperidin has been investigates in an EPISKIN test for skin corrosion irritation as well as in a BCOP test for eye corrosion followed by an eye-irritation in-vivo test for eye irritancy. All tests turned out negative and as a result hesperidin is not subject to classification accoring to CLP (Regulation (EC) No 1272/2008) and/or DSD (Directive 67/548/EEC).