Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3700 AV Zeist, The Netherlands

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Celitement
- Chemical name: Calcium hydrosilicate
- Physical state: White powder
- pH-value: 10.5
- Expiration date of the lot/batch: 12/2015
- Stability under test conditions: Hydration reaction with water
- Storage condition of test material: Room temperature protected from light, in original container in evacuated desiccator

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor

Test animals

Details on test animals and environmental conditions:
The EpiDerm™ (EPI-200) skin model consisted of normal human epidermal keratinocytes from one single donor, derived from neonatal-foreskin tissue. The keratinocytes were plated on chemically modified, collagen-coated, 9 mm ID cell culture inserts (surface area 0.64 cm2). The skin models are commercially available and were obtained from MatTek In Vitro Life Science Laboratories (IVLSL), Slovakia.

Test system

Type of coverage:
other: EpiDerm™ (EPI-200) skin model
Vehicle:
unchanged (no vehicle)
Controls:
other: positive controls: Skin corrosion test, 8 M potassium hydroxide solution and skin irritation test: 5% aqueous sodium dodecyl sulphate (SDS). Negative controls, skin corrosion test:Milli-Q water and skin irritation test:phosphate buffered saline (PBS).
Amount / concentration applied:
25 ± 1 mg
Duration of treatment / exposure:
In the in vitro skin corrosion test, the skin membranes were topically exposed to the undiluted test substance for 3 min and 60 min.
In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min
Observation period:
Skin corrosion test: Immediately after exposure the viability of the epidermal cells was assessed using the MTT test.
Skin irritation test: Viability of the epidermal cells was assessed using the MTT test after 42 h of culture.
Details on study design:
Experimental procedures
- Preliminary tests: Some chemicals are known to non-specifically reduce MTT, resulting in a blue precipitate. Therefore, ca. 25 mg of the test substance was incubated in 1 mL of a 1 mg/mL MTT solution for 187 min at ca. 37°C and 5% CO2 and the formation of a blue formazan product was assessed visually. At the end of the incubation period the MTT solution had turned blue/purple, indicating that the test substance has the potential to reduce MTT. Therefore, additional checks were performed in the main study using freeze-killed skin models (frozen controls). The test substance and negative control were applied to frozen controls (n=2 per test group, for both the in vitro skin corrosion and irritation test).
- Main tests: Two tests were perfromed: One test group (group B) in which the skin surface was moistened prior to application (according to OECD guidelines 431 and 439, to improve tissue surface contact with the test substance) and one test group (group A) in which the skin surface was not moistened prior to application (because it was known that the test substance reacts with an aqueous solution.
- Exposure to study substances: For the in vitro skin corrosion test, the skin models were topically exposed to 50 μL of the negative control (Milli-Q water) or positive control (8 M potassium hydroxide solution), or 25 ± 1 mg of the test substance, at the end of the acclimatization period. Prior to exposure of group B, the skin surface was moistened with 25 μL Milli-Q to improve tissue surface contact with the test substance. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of the liquid study substances. Exposure for 3 min was performed at ambient temperature; exposure for 60 min was initiated at room temperature, followed by incubation in a humidified incubator at ca. 37°C and 5% CO2. Frozen controls were included for both the 60 min and 3 min exposure period. After completion of each exposure period, the skin models were removed from the well and carefully washed using an excess of PBS. The inserts were stored in assay medium until all skin membranes had been washed. Subsequently, viability was determined using the MTT assay.
- Exposure to study substances: For the in vitro skin irritation test, the skin models were topically exposed to 30 μL of the negative control (phosphate buffered saline) or positive control (5% aqueous sodium dodecyl sulphate), or 25 ± 1 mg of the test substance, at the end of the second acclimatization period. Prior to exposure of group B, the skin surface was moistened with 25 μL PBS to improve tissue surface contact with the test substance. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of the liquid study substances. Exposure was initiated at ambient temperature. Frozen controls were included. After dosing the last skin model, all plates were transferred to a humidified incubator (ca. 37°C and 5% CO2). After 35 min, the plates were removed from the incubator and kept at room temperature until the period of 60 min was completed for the first dosed skin model. After completion of the exposure period, the skin models were removed from the well and carefully washed using excess of PBS. Subsequently, the skin models were blotted dry and carefully dried using a cotton swab. The skin models were transferred to a clean 6-well plate containing fresh medium (0.9 mL/well) and incubated in a humidified incubator (ca. 37°C and ca. 5% CO2). Medium was refreshed after 24 h. Following an additional 18 h incubation period (total post-exposure period of ca. 42 h), viability was determined using the MTT assay.
- MTT test: An MTT solution of 1 mg/mL was freshly prepared by diluting MTT concentrate five times in MTT diluent (provided with the MTT-100 assay kit). The bottom of the skin models was blotted dry and the inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 180 min incubation in a humidified incubator at ca. 37ºC and 5% CO2, the skin models were rinsed three times with PBS. The formazan product was extracted from the skin model using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed at 2-10°C for one day (corrosion) or three days (irritation). Following extraction, the skin models were pierced with a needle to allow the extract to run into the well from which the skin model was taken. The optical density was measured in triplicate in 200 μL sub-fractions using a spectrophotometer set at 570 nm. Extractant solvent alone was used as blank. The mean optical density was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).
 
Interpretation of results:
- Frozen controls: The optical density of the viable skin models was corrected for non-specific MTT reduction of the test substance according to the following formula: True optical density = mean optical density viable skin models treated with the test substance – mean optical density frozen controls, where mean optical density frozen controls = mean optical density of the frozen controls treated with the test substance – the mean optical density of the frozen controls treated with the negative control. If the non-specific reduction of MTT by the test substance was comparable to the negative control, a correction was not required.
- In vitro skin corrosion test: The test was considered valid if the mean optical density of the negative control was ≥ 0.8 and ≤ 2.8, skin models treated with the positive control for 60 min revealed mean tissue viability <15% compared to the negative control, and if the coefficient of variation (CV) calculated from individual skin model viabilities of the two identically treated replicates was ≤ 30% in case the mean tissue viability was between 20% and 100%. The test was considered invalid if the test did not meet one or more of these acceptance criteria. The in vitro corrosion potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model: Mean tissue viability (% of negative control)

Mean tissue viability (% of negative control) Prediction
3 min 60 min
< 50 % - Corrosive (Optional subcategory 1A)
≥ 50 % < 15 % Corrosive (Optional subcategory 1B and 1C)
≥ 50 % ≥ 15 % Non-Corrosive

- In vitro skin irritation test: The test was considered valid if the optical density of the negative control was ≥ 0.8 and ≤ 2.8, if the optical density of the extractant solvent alone was < 0.1, if skin models treated with the positive control demonstrated a mean tissue viability ≤ 20% compared to the negative control, and if the standard deviation (SD) calculated from individual tissue viability percentages of the three replicates was ≤ 18%.The test was considered invalid if the test did not meet one or more of these acceptance criteria. The in vitro irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:

Mean tissue viability (% of negative control) Prediction
Mean tissue viability ≤ 50 % Irritant, UN GHS Category 2
Mean tissue viability > 50 % Non-irritant, UN GHS No Category

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
other: Mean
Time point:
other: 3 min
Score:
92
Remarks on result:
other: Skin corrosion test, 3 min exposure group A.
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
other: Mean
Time point:
other: 3 min
Score:
89
Remarks on result:
other: Skin Corrosion test, 3 min exposure group B.
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
other: Mean
Time point:
other: 60 min
Score:
53
Remarks on result:
other: Skin corrosion test, 60 min exposure group A
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
other: mean
Time point:
other: 60 min
Score:
95
Remarks on result:
other: Skin corrosion test, 60 min exposure group B
Irritation parameter:
other: Tissue viability (% of negative control)
Basis:
other: Mean
Time point:
other: 60 min
Score:
88
Remarks on result:
other: Skin irritation test, 60 min exposure group A
Irritation parameter:
other: Tissue viabillity (% of negative control)
Basis:
other: Mean
Time point:
other: 60 min
Score:
98
Remarks on result:
other: Skin irritation test, 60 min exposure group B
Irritant / corrosive response data:
- In vitro skin corrosion test: Optical density of the negative control (Milli-Q) and the positive control (8 M KOH) demonstrated the expected responses. The CV of those test groups that showed mean viabilities between 20% and 100% was ≤ 30%, except for group A, which showed a CV of 98% after 60 min exposure. Since the mean viability of the replicate with the lowest value was 16% (i.e. > 15%) and indicated a noncorrosive response, the mean viability of group B indicated a non-corrosive response, and the test substance was classified as non-irritant in the in vitro skin irritation test, it was considered not relevant to repeat the test group in the in vitro skin corrosion test, because it would not change the conclusion. Therefore, the study was considered valid. Optical density of the frozen control membranes exposed to the test substance was comparable to the optical density of the frozen control membranes exposed to the negative control. Therefore no data correction was applied.
- In vitro skin irritation test: Optical density of the extractant solvent was within the acceptable range and the negative control (PBS) and the positive control (5% SDS) demonstrated the expected responses. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the study was considered valid. For group A, mean optical density of the viable skin models was corrected for nonspecific MTT reduction of the test substance. Optical density of the frozen control membranes exposed to the test substance was comparable to the optical density of the frozen control membranes exposed to the negative control for group B. Therefore no data correction was applied for group B.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions (OECD 431, 439 and GLP) the test substance is considered to be non-corrosive and non-irritant.
Executive summary:

In accordance to OECD guideline 431, 439 and GLP the test substance was examined for its in vitro skin corrosion and irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin corrosion test, the skin membranes were topically exposed to the undiluted test substance for 3 min and 60 min. Per test group, test was performed in duplicate. Immediately after exposure the viability of the epidermal cells was assessed using the MTT test. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for and 60 min. Per test group, tests was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. In both tests negative and positive controls were run in parallel. Optical density of the test groups was expressed as percentage of the concurrent negative control. Both tests included one test group (group B) for which the skin surface was moistened prior to application (to improve tissue surface contact with the test substance) and one test group (group A) in which the skin surface was not moistened prior to application (because it was known that the test substance reacts with an aqueous solution). In the in vitro skin corrosion test, after 3 min exposure the mean viability of the skin membranes was 92 ± 10% for group A and 89 ± 5% for group B, compared to the concurrent negative control group, respectively. After 60 min exposure, the mean viability of the skin membranes was 53 ± 52% for group A and 95 ± 5 for group B, compared to the concurrent negative control group, respectively. In the in vitro skin irritation test, the mean viability was 88 ± 7% for group A and 98 ± 6% for group B, compared to the concurrent negative control group. Based on the results obtained in the present study, the test substance was classified as non-corrosive and non-irritant