Registration Dossier

Administrative data

Description of key information

Skin corrosion

reconstructed human epidermis (RhE) in vitro model EPISKIN™, OECD 431, GLP, negative

Skin irritation

reconstructed human epidermis (RhE) in vitro model EPISKIN™, OECD 439, GLP, negative

Eye irritation:

bovine cornea ex vivo, OECD 437, GLP, negative

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2017 - Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted on 29 July 2016
Deviations:
yes
Remarks:
please refer to "Principles of method if other than guideline"
Principles of method if other than guideline:
OD values obtained in the main experiment fell out the spectrophotometer linear range, defined by the MTT formazan curve performed on the day of the experiment. As soon as this issue was noted, within the same week of the experiment, a second calibrationcurve was prepared using a higher maximum concentration. Thus, a spectrophotometer linear range including OD values obtained in the main experiment was obtained. No other deviations occurred during the study.
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
test item: treatment level of 20 ± 2mg/epidermis unit, each measuring 0.38cm2 (treatment level: 52.6mg/cm2)
positive and negative control: treatment level of 50 μL/epidermis unit
Duration of treatment / exposure:
exposure period of 3, 60 and 240 minutes
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
40
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
79
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240min
Value:
75
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TP 1646 is identified as non-corrosive to the skin.
Executive summary:

The potential of the test item to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431 and was performed under GLP conditions. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. After addition of the test item to the MTT solution, a blue colour was noted indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay.

In the Main Assay, for each treatment time, the test item (physical state: solid) was applied as supplied in two replicates, at the treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 52.6 mg/cm2). Positive and negative controls (glacial acetic acid and physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability.

The positive control caused the expected cell death (1% of cell viability, when compared to the negative control).

Based on the stated criteria, the assay was regarded as valid.

The NSMTT values were higher than 5%, but lower than 50% at all treatment times, thus these values were subtracted from the concurrent mean OD value of the test item treated tissues, to evaluate the actual viability.

The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TP 1646 is identified as non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2017 - Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted on 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Vehicle:
unchanged (no vehicle)
Details on test system:
EPISKIN™
Commercial Name EPISKIN™ - 0.38 cm2
Supplier SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
Batches 17-EKIN-047 (alive tissues) and 17-EKIN-028 (killed tissues)
Arrived at RTC on 21 November 2017 and 11 July 2017
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
test item: 20±2mg/epidermis unit, each measuring 0.38cm2 (treatment level: 53 μL/cm2)
positive and negative control: 20 μL/epidermis unit
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hour recovery period
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
89
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The mean cell viability of the test item treated tissues, after the blank subtraction, was 89%.
Based on the results obtained, the test item is classified as non-irritant to the skin (UN GHS No Category).
Executive summary:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439 and was performed under GLP conditions. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A blue solution was noted at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential colour interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20±2 mg/epidermis unit, each measuring 0.38cm2 (treatment level: 53 μL/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. Non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.8). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid.

The NSMTT value was −1%, thus only the OD-blank background subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 89% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.6 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item TP 1646 is classified as non-irritant to the skin (UN GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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
Study period:
Sep 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
Version / remarks:
edition adopted 09. Oct. 2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14. Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
Species Bos primigenius Taurus (fresh bovine corneas)

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container.
Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
test item: 20 % in HBSS
negative control: HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20171128
positive control: Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: 20171128
Duration of treatment / exposure:
4 hours at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1 °C
Number of animals or in vitro replicates:
3
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
2.78
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
0.56
Vehicle controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
0.97
Vehicle controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The test item showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.44.
Executive summary:

This in vitro study, according to OECD guideline 437 and EU Method B.47, under GLP-conditions, was performed to assess corneal damage potential of the test item by quantitative measurements of changes in opacity and permeability in a bovine cornea.

Two experiments were performed. The first experiment was considered invalid because all of the three replicates caused different classifications. The first experiment is not reported but the raw data are kept in the GLP-archive of the test facility. The second experiment was considered as valid. Only the second experiment is reported here. The test item was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The test item was tested as 20% solution in HBSS. Under the conditions of this test, the test item showed no effects on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 1.44. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage. The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

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

Additional information

Skin corrosion

The potential of the test item to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431 and was performed under GLP conditions. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

A preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. After addition of the test item to the MTT solution, a blue colour was noted indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay.

In the Main Assay, for each treatment time, the test item (physical state: solid) was applied as supplied in two replicates, at the treatment level of 20 ± 2 mg/epidermis unit, each measuring 0.38 cm2(treatment level: 52.6 mg/cm2). Positive and negative controls (glacial acetic acid and physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included for each treatment time.

In the Main Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value of the treatment series and thus represents 100% of cell viability.

The positive control caused the expected cell death (1% of cell viability, when compared to the negative control).

Based on the stated criteria, the assay was regarded as valid.

The NSMTT values were higher than 5%, but lower than 50% at all treatment times, thus these values were subtracted from the concurrent mean OD value of the test item treated tissues, to evaluate the actual viability.

The mean cell viability of the test item treated tissues, after the blank subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TP 1646 is identified as non-corrosive to the skin.

Skin irritation

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439 and was performed under GLP conditions. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A blue solution was noted at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless solution was observed, indicating that the test item has no potential colour interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay. In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20±2 mg/epidermis unit, each measuring 0.38cm2(treatment level: 53 μL/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. Non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.8). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid. The NSMTT value was −1%, thus only the OD-blank background subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 89% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.6 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item TP 1646 is classified as non-irritant to the skin (UN GHS No Category).

Eye irritation:

This in vitro study, according to OECD guideline 437 and EU Method B.47, under GLP-conditions, was performed to assess corneal damage potential of the test item by quantitative measurements of changes in opacity and permeability in a bovine cornea. Two experiments were performed. The first experiment was considered invalid because all of the three replicates caused different classifications. The first experiment is not reported but the raw data are kept in the GLP-archive of the test facility. The second experiment was considered as valid. Only the second experiment is reported here. The test item was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The test item was tested as 20% solution in HBSS. Under the conditions of this test, the test item showed no effects on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 1.44. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage. The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Justification for classification or non-classification

Based on the reliable test results the substance is not classified according to Regulation (EC) No 1272/2008.