Reaction mass of 1,1-bis(phenethyloxy)ethane and [2-(1-ethoxyethoxy)ethyl]benzene

Endpoint summary

Description of key information

The substance is irritating to the skin but not to the eye.

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

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Physical Appearance: Colourless liquid
- Date of Expiry: 12/07/2018
- Storage Condition: Ambient (21 to 29°C)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM: The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) was used as test system provided by MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic
PREPARATION OF CHEMICALS, REAGENTS AND MEDIA: MTT solution was prepared by thawing the MTT concentrate (MTT-100-CON). MTT concentrate of 2 mL (5 mg/mL) was diluted to 10 mL by adding 8 mL of MTT diluent (MTT-100-DIL) to obtain the final concentration of 1 mg/mL. As the MTT is light sensitive MTT solution was stored at 5±3°C in a vial wrapped with aluminum foil. The sterile Dulbecco’s Phosphate Buffered Saline (DPBS) provided by MatTek was used as negative control. Ready to use 5% Aqueous Sodium Dodecyl Sulfate (SDS) provided by MatTek was used as positive control (PC).
EXPERIMENTAL PROCEDURE
- Test for Mesh Compatibility: For even spreading of liquid test items, a nylon mesh was used. The compatibility of liquid test items with the nylon mesh was checked by adding 30 μL of test item onto the nylon mesh placed on a glass slide. After 60 minutes of incubation, the mesh was observed under a microscope. As the nylon mesh texture was unaltered, a nylon mesh was used for spreading of the test item onto the tissues.
- Test for Interference of Test Item with MTT Endpoint and Correction Procedures: To a glass test tube containing 300 μL of distilled/deionized water and 300 μL of isopropanol, 30 μL of the test item was added. The mixture was incubated in the incubator (37±1°C, 5±1% CO2) for approximately 60 minutes. At the end of the exposure time, the mixture was mixed thoroughly and evaluated for the presence and intensity of the staining. As there was no color change observed after incubation, further testing in was not performed. In addition, the test item was evaluated for its potential to interfere with the MTT assay. To test if a material directly reduces MTT, 30 μL of the test item was added to 1 mL of the MTT medium in a 6-well plate and incubated in the incubator (37±1°C, 5±1% CO2) for 60 minutes. The MTT solution containing the test item did not turn to a blue/purple color, hence the test material is considered as a non-reducer of MTT. The additional functional check was not performed.
- Main test: Upon receipt of the shipment, all kit components were examined for integrity. All information about the supplied material was recorded and documented. The MTT diluent and vial containing the MTT concentrate were stored in the refrigerator (2 to 8°C) and in the deep freezer (-20±5°C), respectively. The assay medium was allowed to reach room temperature. To each well of four sterile 6-well plates, 0.9 mL of the assay medium was added. (One 6-well plate for pre-incubation of three inserts). The plastic bag containing the 24-well plate with epidermal tissues was opened in the bio safety cabinet. The sterile gauze was removed and each insert containing the epidermal tissue was carefully taken out using sterile forceps. The remaining agarose that adhered to the outer sides of the insert was removed by gentle blotting on the sterile filter paper, and tissues were placed in the empty, sterile 24-well plate. The inserts were inspected visually within 5 minutes and all the tissues were used for the experiment as they were found without any defects or excess moisture on the surface. Tissues were transferred to a 6-well plate pre- filled with 0.9 mL of assay medium. Plates were placed in a CO2 incubator (37±1°C, 5±1% CO2) overnight for 15 hours and 50 minutes. The upper row of four 6-well plates were pre-filled with 0.9 mL of assay medium and each 6-well plate was used for one treatment. Approximately five minutes before exposure with the test item, the incubated tissues were removed from the CO2 incubator and the surface of the tissues were evaluated. All the tissues used for the treatment were without moisture or any visible defect. Hence, all tissues were used for testing. Each 6-well plate lid was labeled with the treatment code. Test item, positive control and negative control were tested in triplicate. A quantity of 30 μL of DPBS (NC) and 5% SDS (PC) were dispensed directly atop the tissue at 1-minute intervals to facilitate rinsing of the NC and PC after exposure. The tissues were exposed to 30 μL of test item. A nylon mesh was placed on the treated tissues for even spreading of test item. The plates with treated tissues were kept in the sterile hood, until the last tissue was dosed. All plates were transferred to the humidified CO2 incubator (37±1°C, 5±1% CO2) for 35 minutes. After 35 minutes of incubation, all plates were removed from the incubator and placed inside the sterile hood and waited until the period of 60 minutes was completed for the first treated tissue. After 60±1 minutes of test item exposure, the tissues were rinsed with sterile DPBS by filling and emptying the tissue insert for 15 times to remove any residual test item. The constant stream of DPBS was applied from the nearest distance from the tissue surface. After the 15th rinse with washing bottle, the inserts were completely submerged 3 times in approximately 50 mL of DPBS and shake to remove all traces of test item/NC/PC. Finally, each tissue was rinsed once from the inside and once from the outside with sterile DPBS. The excess of DPBS was removed by gentle shaking the insert and blotting the insert on sterile blotting paper. Blotted tissue inserts were transferred to new 6 well plates pre-filled with fresh assay medium. The tissues were incubated in the CO2 incubator (37±1°C, 5±1% CO2) for the next 24 hours and 10 minutes. After 24 hours and 10 minutes of incubation, inserts were transferred into the lower part of the 6-well plate, pre- filled with 0.9 mL of media and continued with the post-incubation (37±1°C, 5±1% CO2) for another 20 hours and 40 minutes. Two 24-well plates were labeled with the treatment code. One plate was used for tissue incubation with MTT and the other for the extraction step. The MTT solution was prepared by thawing the MTT concentrate (5 mg/mL) and diluting 1 mL to 5 mL with the MTT diluent. Final concentration of MTT solution was 1 mg/mL. A volume of 300 μL of MTT solution was added into each well of the 24-well plates. The 6-well plate was removed from the incubator, the bottom of the inserts was blotted on a blotting paper, and transferred into the 24-well plate pre- filled with MTT. The 24-well plate was placed in the CO2 incubator (37±1°C, 5±1% CO2) for 3 hours ±5 minutes. After a post incubation of the inserts for 3 hours, the MTT medium was gently aspirated from all the wells using a syringe. The wells were refilled with DPBS and aspirated again. This rinsing was repeated two times to ensure that tissues were dry after the last aspiration. After the washes, the inserts were transferred into a new 24-well plate. The inserts were immersed by adding 2 mL of extractant solution (MTT-100-EXT) into each insert. The level raised above the upper edges of the insert, thus completely submerging the tissues. The 24-well plate was sealed with parafilm to inhibit extractant evaporation. The MTT was extracted from the tissues for 2 hours at room temperature with gentle shaking on a plate shaker (~ 120 rpm). After completion of the extraction period, the inserts were pierced with an injection needle and allowed the extract to run into the well from which the insert was taken. The punctured insert was discarded and the solution in the well was mixed three times using a pipette until it became homogenous. For each tissue, two 200 μL aliquots of the purple formazan solution were transferred into a 96-well flat bottom microtiter plate. Extractant solution was used as blank. The optical density (OD) of the MTT extracts in a 96-well plate was read in a plate reader using a wavelength of 570 nm. Results were entered into the spreadsheet provided by MatTek for automatic calculation of the results.
TEST ACCEPTANCE CRITERIA: (1) The assay met the acceptance criterion as the mean OD570 of the NC tissues is 1.794 which is in the range of ≥0.8 and ≤ 2.8. (2) The assay met the acceptance criterion as the mean viability of PC tissues is 10.4% which is ≤ 20% of the negative control tissues and the SD of the three tissues replicates is 0.26 (below the 18%). (3). The assay met the acceptance criterion as the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is <18% i.e., in the range of 0.26 to 2.07.
EVALUATION AND INTERPRETATION OF RESULTS: A test item is considered to be “irritant” to skin in accordance with UN GHS Category 2, if the tissue viability after exposure and post-treatment incubation is ≤ 50%. The test item is considered as non- irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is > 50%.

Irritation / corrosion parameter:

% tissue viability

Value:

11.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The mean OD of the negative control tissues was 1.794 which is within the range of ≥0.8 and ≤2.8, hence the tissues were considered as viable after shipping and storing procedures and under specific conditions of use. The percentage viability of the tissues was measured by performing the MTT assay after 60 minutes of treatment and a post incubation period of 44 hours and 50 minutes. The mean percentage viability of with the test item treated tissues was 10.6 which is <50% of the negative control, hence the test item is considered as irritant whereas themean percentage viability of the with the positive control treated tissues was 10.4 which is <50% of the negative control, which clearly represents the irritation potential of the PC.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes

Specific details on test material used for the study:

- Physical Appearance: Colourless liquid
- Date of Expiry: 12/07/2018
- Storage Condition: Ambient (21 to 29°C)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house.
- Number of animals: 10
- Characteristics of donor animals: Age of cattle : 3.8 to 4.5 years, 5 males and 5 females
- Storage, temperature and transport conditions of ocular tissue: The head of the cattle were not rinsed with detergent. Eyes were enucleated as soon as possible after death and were immersed in Hank’s Balanced Salt Solution (HBSS) with antibiotics (Penicillin and Streptomycin), placed in a suitable container and transported to the test facility in cool packs.
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the test facility, eyes were examined for defects including opacity, scratches and no vascularization. Only corneas free of such defects were used in the experiment. Horizontal diameter and central corneal thickness (CCT) was measured. Cornea with horizontal diameter <28.5 mm and CCT <900 μm were selected.

Details on study design:

PROCEDURE
- Preparation of Eyes: Before the start of the experiment, the opacity of empty cornea holders filled with pre-warmed EMEM was measured and the mean opacity value obtained was considered as I0. Corneas free of defects were dissected with 2 to 3 mm rim of sclera remaining. Isolated corneas were mounted in designated corneal holders by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both the chambers were joined together by tightening the chamber screws. The corneal holders were equilibrated for at least 1 hour by allowing the corneas to equilibrate and to achieve normal metabolic activity with pre-warmed and red-free EMEM supplemented with 1% Fetal
- Bovine Serum and 1% antibiotics - Penicillin and Streptomycin in an incubator at 32±1°C. Fresh pre-warmed and red-free EMEM was added to both chambers of the cornea holders after completion of the equilibrium period and baseline opacity was recorded for each cornea. The opacity of each cornea was calculated by using the formula I0/I and the opacity value was calculated for initial readouts (Before Treatment) by using the formula [(I0/I-b)/a] where a=0.0251, b=0.9894. Corneas lesser than 7 opacity units, after an initial 1-hour equilibration period, were used for the experiment.
- Application of the Test item: The medium from the anterior chamber was removed and the test item was added by following the Close Chamber Method. A quantity of 750 μL of test item was introduced into the anterior chamber through the dosing hole of the corneal holder and the holes were sealed during exposure and the holders were incubated horizontally at 32±1°C. The corneas were exposed to the test item for approximately 10 minutes. Similar procedure was followed for the negative and positive controls. Three corneas per group were used.
- Post Exposure: After the exposure period, the test item, negative and positive controls were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red till no visual evidence of the test item was observed. Finally, the corneas were rinsed with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red and incubated at 32±1°C for 2 hours. Post exposure corneas were observed visually for tissue peeling, residual test chemical, and non-uniform opacity patterns.
- Measurements of End Points: After a post-incubation period of 2 hours, the opacity was measured with the aid of an opacitometer. The permeability was determined by adding 1 mL of 4 mg/mL of sodium fluorescein to the anterior chamber of the cornea holder, while the posterior chamber was filled with fresh MEM. The holders were incubated in a horizontal position for 90 min at 32±1°C. The permeability was measured with the aid of a spectrophotometry as optical density at 490 nm.
- Histopathological Evaluation: Corneas were preserved in 10% neutral buffered formalin for further histopathological examination. All the corneas including the negative control, positive control and test item were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin and subjected to histopathological evaluation.

DATA EVALUATION
- Data Derivation: The opacity and mean permeability values were corrected for background opacity and permeability at OD490 values using the following formula: True value of opacity of positive control/test item = Change in opacity value of positive control/test item - mean change in opacity value of negative control. True value of optical density at 490 nm of positive control/test item = OD of positive control/test item - OD of negative control. The mean opacity and permeability at OD490 values for each treatment group was combined and in vitro Irritancy Score (IVIS) for each group was derived using following formula. IVIS= Mean Opacity value + (15 × Mean permeability OD490 value).
- Decision Criteria: Test item with a IVIS score ≤ 3 would be considered as UN GHS no category. Test item with a IVIS score >3 and ≤ 55 would be considered as: “no prediction can be made”, subsequently testing with any other adequate method remains at the discretion of the sponsor. Test item with a IVIS score > 55 would be considered as severe irritant causing serious eye damage and classified as UN GHS category 1 without further testing.
- Study Acceptance Criteria: The study was accepted: As the negative control response resulted in opacity and permeability values less than the established upper limits of background opacity and permeability values for bovine corneas treated with the respective negative control. As the positive control gave an IVIS that falls within two standard deviations of the historical mean.

Irritation parameter:

in vitro irritation score

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- In vitro Irritancy Score (IVIS): The mean corrected opacity and mean corrected permeability values of the test item are 0.59 and -0.007, respectively. The in vitro Irritancy Score (IVIS) of the test item is 0.5 which is considered as UN GHS no category. Treatment with the positive control resulted in mean corrected opacity and mean corrected permeability values of 92.79 and 0.829, respectively. The in vitro Irritancy Score (IVIS) of the positive control was determined to be 105.2, indicating corrosivity or severe irritancy to Bovine corneas.
- Histopathological Evaluation: In the histopathological examination, the corneas treated with the negative control and test item did not show any abnormalities. In the positive control treated corneas, histopathology data showed moderate diffuse cytoplasmic and nuclear vacuolization in the wing and basal layers of the epithelium. In all the corneas, multifocal minimal expansion of the superficial collagen was observed in stroma. Endothelium was apparently normal in all three corneas.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation

No in vitro skin corrosion test has been performed as in the acute dermal toxicity study no corrosive effects were observed.

Skin irritation was investigated in an in vitro skin irritation test using the Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) according to OECD Guideline 439 and following GLP. The test item did not develop any colour when dissolved in distilled water and is considered as a non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution. After receipt of the tissues, a visual inspection was performed to verify the defects. As there were no tissue defects, air bubbles or excess moisture observed, all the tissue inserts were used for the study. Tissue inserts were transferred to the upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in a CO2 incubator for 15 hours and 50 minutes. After the incubation period, tissues were topically exposed to 30 μL each of DPBS (negative control), 5% aq. SDS solution (positive control), or test item. All the treatments were performed in triplicate. After 60 minutes of exposure with the negative control, positive control and test item, the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in a CO2 incubator for 24 hours and 10 minutes. After the incubation period, tissue inserts were shifted from the upper wells to the lower wells prefilled with 0.9 mL of assay medium. After the media change, the tissues were incubated for an additional 20 hours and 40 minutes in a CO2 incubator. After the incubation period, the bottom of the tissue inserts were blotted and transferred into MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours and 20 minutes using extraction solvent (MTT-EXT-100). The optical density of the extracted formazan was measured in a 96-well plate spectrophotometer at 570 nm. The viability of the tissues was calculated by entering OD values in the spread sheet provided by MatTek. The percentage viability of the negative control, positive control and test item was 100±2.07, 10.4±0.26, and 10.6±0.32, respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as irritant. Similarly the percentage viability of the positive control is less than 50% of the negative control, which clearly represents the irritation potential of positive control. Based on the results obtained under the laboratory testing conditions, the test item has been categorized as irritant in accordance with UN GHS Category 2, as the mean percentage tissue viability was not greater than 50% of the negative control.

Eye irritation

Ocular corrosivity or severe irritancy of the test substance was investigated according to OECD Guideline 437 and following GLP. Eyes of cattle were collected from a slaughter house. Eyes were immersed in Hank’s Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) and placed in a suitable container and transported to the test facility on cool packs. Eye balls free of defects were selected for the experiment. The opacity of empty cornea holders filled with pre-warmed EMEM was measured and the mean opacity values obtained were determined as I0. Cornea holders with selected corneas were equilibrated at 32±1°C for 1 hour with EMEM supplemented with 1% Fetal Bovine Serum and 1% antibiotics. The baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed among the treatment groups. A quantity of 750 μL of test item, distilled water (negative control), and ethanol (positive control) was introduced in triplicate into the anterior chamber of the designated cornea holders and incubated at 32±1°C for 10 minutes. Treated corneas were washed with EMEM with phenol red. Opacity was measured with the aid of an opacitometer. Permeability was determined spectrophotometrically, after an incubation period of 90 min at 32±1°C, at 490 nm (OD490) using 4 mg/mL sodium fluorescein. Baseline opacity and permeability values obtained for negative control (distilled water) treated corneas were used for correction. The mean corrected opacity and mean corrected permeability values of the test item were 0.59 and -0.007, respectively. The in vitro Irritancy Score (IVIS) of the test item was determined to be 0.5. Treatment with the positive control resulted in mean corrected opacity and mean corrected permeability values of 92.79 and 0.829, respectively. The IVIS of the positive control was determined to be 105.2, indicating corrosivity or severe irritancy to Bovine corneas. In the histopathological examination, the corneas treated with the negative control and test item did not show any abnormalities. In the positive control treated corneas, histopathology data showed moderate diffuse cytoplasmic and nuclear vacuolization in the wing and basal layers of the epithelium. In all the corneas, stroma was observed with multifocal minimal expansion of the superficial collagen. Endothelium was apparently normal in all three corneas. Based on the results obtained in the Bovine Corneal Opacity Test, the test item induced an IVIS of 0.5 after 10 minutes of treatment. There was no appreciable increase in both the endpoints “permeability” as well as “opacity”. As the test item induced an IVIS ≤3, it is considered as UN GHS no category.

Justification for classification or non-classification

The test substance has to be classified as Skin Irrit 2: H315: Causes skin irritation according to Regulation (EC) No 1272/2008. The test substance does not have to be classified for eye irritation according to Regulation (EC) No 1272/2008.