Registration Dossier

Administrative data

Description of key information

The substance is irritating to the skin, but not irritating 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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Physical Appearance: Almost colourless to yellow-brown liquid
- Date of Expiry: 01-08-2018
- 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 16 hours and 4 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 5 minutes. After 24 hours and 5 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 1.462 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 8.3% which is ≤ 20% of the negative control tissues and the SD of the three tissues replicates is 0.35 (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 0.35 to 12.49.
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:
6.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
The mean OD of the negative control tissues was 1.462 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 45 minutes. The mean percentage viability of with the test item treated tissues was 6.8 which is <50% of the negative control, hence the test item is considered as irritant whereas the mean percentage viability of the with the positive control treated tissues was 8.3 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

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1981
Deviations:
yes
Remarks:
no details on purity, no details on animal housing and environmental conditions.
Principles of method if other than guideline:
Modified Federal Hazardous Substances Labelling Act Method.
GLP compliance:
no
Test material information:
Composition 1
Specific details on test material used for the study:
Appearance: liquid
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Unilever Environmental Safety Division, Colworth Laboratory
- Age at study initiation: 7 weeks
- Weight at study initiation: 1.9 - 2.0 kg
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
0.1 mL
Duration of treatment / exposure:
Single instillation on Day 1
Observation period (in vivo):
5 days
Number of animals or in vitro replicates:
2 females and 1 male
Details on study design:
STUDY DESIGN
The test material was applied undiluted in 3 animals.

TREATMENT
The substance was applied to one eye of the rabbits by gently pulling the lower lid away from the eye ball and placing 0.1 mL in the sac.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Irritation: The eyes were examined 24 hours after treatment and thereafter at daily intervals and graded for corneal, conjunctival and iridial damage. Eyes were examined before application of materials and at daily intervals afterwards with a slit lamp and corneal swelling is measured. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405 (1981).
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.17
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal: #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The substance caused slight to moderate corneal opacities in all three animals, with conjunctival swelling in two animals. All three animals had slight conjunctivitis. The observed effects were fully reversible within 4 days.
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.17
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal: #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
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 16 hours and 4 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±12.49, 8.3±0.35, and 6.8±0.83, 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

For the determination of the eye irritating properties of the substance to be registered, data on a structural analogue: 2 -heptylcyclopentanone was used. The justification for the use of data on 2 -heptylcyclopentanone is included in the dossier. 2 -Heptylcyclopentanone was tested in an eye irritation test in two female and one male rabbit according to a method equivalent to the OECD 405 guideline. The substance caused slight to moderate corneal opacities in all three animals, with conjunctival swelling in two animals. All three animals had slight conjunctivitis. The observed effects were fully reversible within 4 days. The results showed that 2 -heptylcyclopentanone is not considered to be an eye irritant. Subsequently, the substance to be registered is also not considered to be an eye irritant.

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 does not have to be classified as eye irritant according to Regulation (EC) No 1272/2008.