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Administrative data

Description of key information

The substance was shown to be non-irritant to skin and non-irritant to eyes. Data on respiratory irritation are not available.

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:
14.05.2012 - 21.08.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study performed according to GLP
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Species:
other: seeded human epidermal keratinocytes
Strain:
other: supplied by SkinEthic Laboratories, Lyon, France
Details on test animals 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
Vehicle:
unchanged (no vehicle)
Controls:
other: sodium dodecyl sulphate (SDS) 5% as positive control
Amount / concentration applied:
10 µL of the undiluted test item were applied to each of triplicate tissues.
For the positive and negative controls also each 10 µL were dosed per tissue.
Duration of treatment / exposure:
The negative and positive control, and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues. The 12-well plates were placed into the incubator for 15 min at 37 ±1.5 °C, 5 ±0.5% CO2 .
Observation period:
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for approximately 42 hours at 37 ±1.5 °C, 5 ±0.5% CO2 .
Number of animals:
not relevant, measurements were made in triplicates for negative control, positive control and test item.
Details on study design:
The MTT concentrate was thawed on the day of testing and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO 2 ) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol / 2 N HCl 49:1 (v/v)) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 72 hours in the refrigerator.
Per each tissue sample 2 x 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax ® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the medium beneath the tissues was replaced before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". MTT reducing capability of the test item was tested as described in section “9.5 Test for Direct MTT Reduction”.
Irritation / corrosion parameter:
other: other: Relative absorbance (% of negative control)
Value:
108.9
Remarks on result:
other:
Remarks:
Basis: other: absorbance of negative control. Time point: 6 days following incubation. Max. score: 100.0. Reversibility: other: not applicable. (migrated information)

Quality criteria

The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability meets the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5. The standard deviations in between tissues of the same treatment group should be ≤ 18%. An assay meets the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%. The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (the acceptance limit of the IC 50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS). The historical data (means, standard deviation, and ranges) of the positive control as well as for the negative control obtained with the EpiSkin™ model at Harlan CCR are mentioned in the report.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be Non-Irritant (NI) in this human epidermis skin model test.
Executive summary:

The relative mean absorbance of the test item treated tissues was 108.9% (relative to negative control) after a 15-Minute exposure period. Thus, the test substance 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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1989 - 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP, reliability reduced from 1 to 2 as study is used for read-across
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
The testing was made on three SPF albino female rabbits of the stock Mol: Russian from Møllegaard Breeding Centre ApS, Ejby, DK-4623 LL. Skensved. The rabbits weighed 2300-2600 g and were earmarked with National Wing Bands, During a pre-period of one week and throughout the experiment the rabbits were kept to single PPL cages, 45 × 55 cm, with perforated floor. The rabbits had free access to food pellets "Altromin 2123" from Chr. Petersen A/S, DK-4100 Ringsted and to fresh water. The room temperature was 21 ± 2 °C and the relative humidity 55 ±15%. The air was changed 6 times an hour, and light was on from 6 to 18 hrs. Declaration and analysis certificate of diet mixture weere documented. The rabbits were kept in animal room No. 1.
Vehicle:
other: Diethyl phthalate (DEP)
Controls:
other: right eyes remained untreated and served as control
Amount / concentration applied:
50% (w/w)
Duration of treatment / exposure:
one hour (eyes were rinsed with 20 ml 0.9% sodium chloride solution after 1 hour examination)
Observation period (in vivo):
1, 24, 48 and 72 hours after the treatment
Number of animals or in vitro replicates:
3 females
Details on study design:
The day before testing both eyes of the animals were examined with an ophthalmoscope to ensure there were no eye defects or irritation. The examination was performed before and after instillation of oculogottae floureaceini (Ph.N,).
Only the left eyes were treated. The right eyes remained untreated and served as control.
About 0.1 ml of the diluted test article was placed in the left eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was dropped. The lids were then gently held together for about one second. The eyes were examined and the grade of ocular reaction was recorded 1 hour later. 24 hour later an examination was performed before and after installation of oculoguttao flouresceini. After the examined the eyes were rinsed with 20 ml of a 0.9% sodium chloride solution.The eyes were also examined 48 and 72 hours after the treatment
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24-72hours
Score:
0
Max. score:
4
Remarks on result:
other: no positive reaction at any tested group
Irritation parameter:
iris score
Basis:
mean
Remarks:
for all animals
Time point:
other: 24-72hours
Score:
0
Max. score:
2
Remarks on result:
other: no positive response in all animals
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24-72hours
Score:
0.1
Max. score:
3
Reversibility:
fully reversible within: 72hours
Remarks on result:
other: Slight reactions of conjunctiva (redness) were seen in one rabbit at the 1- and 24-hour reading No reactions of the conjunctiva, iris or cornea were observed in any of the rabbits at the 48- and 72-hour readings.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24-72hours
Score:
0
Max. score:
4
Remarks on result:
other: no positive response in all animals
Irritant / corrosive response data:
Slight reactions of conjunctiva (redness) were seen in one rabbit at the 1- and 24-hour reading. No reactions of the conjunctiva, iris or cornea were observed in any of the rabbits at the 48- and 72-hour readings.
Other effects:
no data
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental condition, it can be concluded that 2-ethylhexyl salicylate in a 50% solution caused no eye irritation to rabbits.
Executive summary:

The primary eye irritant effect of 2-ethylhexyl salicylate in a 50% solution was investigated according to the method recommended in the OECD Guideline No. 405"Acute Eye Irritation/Corrosion". About 0.1 ml of the diluted test article was placed in the left eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup into which tho test substance was dropped. The lids were then gently held together for about one second. The eyes were examined and the grade of ocular reaction was recorded 1 hour later. 24 hour later an examination was performed before and after installation of oculoguttao flouresceini. After the examined the eyes were rinsed with 20 ml of a 0.9% sodium chloride solution.The eyes were also examined 48 and 72 hours after the treatment. Slight reactions of conjunctiva (redness) were seen in one rabbit at the 1- and 24-hour reading. No reactions of the conjunctiva, iris or cornea were observed in any of the rabbits at the 48- and 72-hour readings.

Based on the description as above, test article in 50% solution did not cause positive eye response in rabbits.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

The reference substance has been investigated in a human skin model test (in vitro, EPISKIN).

The relative mean absorbance of the test item treated tissues was 108.9% (relative to negative control) after a 15 -minute exposure period. Thus, the test substance had no effect on viability and the substance is considered non-irritant. A supporting study investigating a skin formulation containing 10 - 15% reference substance to human volunteers also showed no irritation marks.

Eye irritation / damage

The primary eye irritant effect of 2-ethylhexyl salicylate (structural surrogate to homosalate having comparable phys-chem properties and the same structural elements - only the alcoholic alkyl moiety is slightly different) in a 50% solution was investigated according to the method recommended in the OECD guideline No. 405 "Acute Eye Irritation/Corrosion".About 0.1 ml of the diluted test article was placed in the left eye of each rabbit by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was dropped. The lids were then gently held together for about one second. The eyes were examined and the grade of ocular reaction was recorded 1 hour later. 24 hour later an examination was performed before and after installation of oculoguttao flouresceini. After the examined the eyes were rinsed with 20 ml of a 0.9% sodium chloride solution.The eyes were also examined 48 and 72 hours after the treatment. Slight reactions of conjunctiva (redness) were seen in one rabbit at the 1- and 24-hour reading. No reactions of the conjunctiva, iris or cornea were observed in any of the rabbits at the 48- and 72-hour readings. Based on the description as above, test article in 50% solution did not cause positive eye response in rabbits.

In a supporting study the potential irritant effect of a sunscreen containing 12% homosalate was investigated in 3 male New Zealand White rabbits. 0.1 ml of the unchanged sunscreen was placed into the conjunctival sac of the right eye of each of the animal. The left eye was not treated and served as control. The test substance was not washed out. Thereafter, the eyes were examined with the aid of an auxiliary light source for signs of irritation covering the cornea, iris and the conjunctiva of each animal at 1, 24, 48 and 72 hours. The findings were scored according to Draize et al. (1959). Treatment had no effect on the cornea of the rabbits at any time-point. Exposure to the sunscreen resulted in iritis in 2/3 rabbits at the 1-hour scoring interval but recovered completely within 24 hours. Conjunctivitis consisting of redness, swelling and/or discharge was recorded in all rabbits at the 1-hour interval, improved in incidence and severity subsequently. At 72 hours no conjunctival reaction was noted. Therefore, it was concluded that the sunscreen containing 12% homosalate formulation led to only slight eye irritation under the conditions of the study.

 


Justification for selection of skin irritation / corrosion endpoint:
Valid OECD guideline study performed under GLP

Justification for selection of eye irritation endpoint:
Valid OECD guideline study on surrogate substance downgraded to Reliability 2 due to read-across.

Justification for classification or non-classification

The reference substance was investigated for skin irritation/corrosion in a human skin model test (EPISKIN) and was found negative of skin irritation/corrosion. A structural surrogate was investigated for eye irritation and found negative, supported by an in-vivo eye irritation/corrosion test of a formulation containing 12% of the reference substance which only showed slight irritation at beginning of the test. Thus, the substance is not classifiable for skin and eye irritation/corrosion according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC). Data on respiratory irritation are not available and due to lack of data the substance is not classified for respiratory irritation.