Registration Dossier

Administrative data

Description of key information

No skin irritating effects were observed in in vitro test or the dermal toxicity study in rats.

The BCOP assay showed that the test item is not highly corrosive, but is not eligible to give a clearly negative result for eye irritation.

The in vivo eye irritation study in rabbits showed that the test substance has no significant effect on the eye of the rabbit.

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:
25 November 2014 to 09 February 2015
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Details on test animals and environmental conditions:
Test item: EPISKIN™ - 0.38 cm2.
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon – France).
Batch numbers: 15-EKIN-005 (alive tissue), 14-EKIN-037 and 14-EKIN-048 (killed tissues).
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2).
Duration of treatment / exposure:
Exposure period of 15 ± 0.5 minutes minutes followed by a 42 ± 1 hours recovery period.
Number of animals:
Number of replicates: 3
Details on study design:
Positive control item: Sodium Dodecyl Sulphate (SDS) (SIGMA, batch 041M8713V), diluted at the final concentration of 5% (w/v) in sterile water for injection (Baxter, batch 13L0503).
Negative control item: D-PBS (GIBCO, batch 1605086).
Non specific colour (NSC) control: test item treated tissues without MTT.
Non specific MTT reduction (NSMTT) control: killed tissues treated with the test item.
Irritation / corrosion parameter:
other:
Value:
98.7
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 h. Max. score: 50.0. Remarks: colorimetric measurement of MTT reduction; Mean relative viability <=50%: Irritant (UNGHS Category 2) Mean relative viability > 50%: Not irritant (UNGHS No Category). (migrated information)
Other effects / acceptance of results:
The Non Specific Colour (NSC), relative to the negative control, was 28.6%; while the non specific MTT reduction (NSMTT) induced by the test item was 18.5%. These results indicated the necessity of further appropriate background subtractions.
Following the appropriate subtractions, a mean cell viability of 98.7%, when compared to the negative control, was obtained with the test item treated tissue. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.9 (lower than 18, as stated in the Study Protocol).
Interpretation of results:
GHS criteria not met
Conclusions:
The test item should be considered not irritant.
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. 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 green coloured suspension was observed at the end of the incubation period, indicating that the test item interacted directly with MTT. Green precipitate was noted. In a second step, the test item was assayed for the ability of colouring water per se; a black suspension was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were 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 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/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. 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 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 (4% of cell viability when compared to the negative control) and variability (SD of % viability equal to 1.1). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤18 ), the assay was regarded as valid.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using one alive treated tissue without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using killed tissues and compared with negative control performed with alive tissues. Based on the results obtained (NSC=28.6% and NSMTT=18.5%), additional background subtractions were performed for the evaluation of irritant properties of the substance. The test item did not induce cell death in any replicate with a mean cell viability of 98.7% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.9 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item is classified as not irritating to the skin.

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:
experimental study
Adequacy of study:
key study
Study period:
From 13. to 17. July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’Arbresle Cedex, France
- Age at study initiation: approximately 16 weeks
- Weight at study initiation: approximately 5 kg
- Housing: 1 per cage (during the study)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C+/-2 °C
- Humidity (%): 55%+/-15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: 13. to 17. July 2015
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:
Only once. Exposure for 1 hour.
No rinsing of the treated eye was performed after dosing.
Observation period (in vivo):
1, 24, 48, 72 hours after dosing.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 1 hour
No rinsing of the treated eye was performed after dosing.

SCORING SYSTEM: Irritation to the cornea, iris and conjunctivae were assigned a numerical value according to the Draize scale.

TOOL USED TO ASSESS SCORE: macroscopic observation.
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No irritation at either the conjunctivae, iris or cornea (score of 0) was recorded in any treated animal, during the whole observation period of the study (72 hours).
Other effects:
There was no indication of a systemic effect.
Blue staining of the fur around the treated eye, due to the colour of the test item, was noted in the animals during the whole observation period.
No signs of pain or distress were observed during the study.
No relevant changes in body weight were seen during the course of the study.
Interpretation of results:
GHS criteria not met
Conclusions:
No signs of irritation were recorded in any treated animal during the observation period.
Executive summary:

The acute eye irritation of the test item was investigated in rabbits.

A 0.1 g aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours after dosing.

No irritation at either the conjunctivae, iris or cornea (score of 0) was recorded in any treated animal, during the whole observation period of the study (72 hours).

There was no indication of a systemic effect.

Blue staining of the fur around the treated eye, due to the colour of the test item, was noted in the animals during the whole observation period.

There were no signs of pain/distress after dosing.

Changes in body weight were not remarkable.

These results indicate that the test item has no irritating effects on the eye of the rabbit.

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would suggest the following:

Classification: No category

Signal word: No signal word required

Hazard statement: No hazard statement required

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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. 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 green coloured suspension was observed at the end of the incubation period, indicating that the test item interacted directly with MTT. Green precipitate was noted. In a second step, the test item was assayed for the ability of colouring water per se; a black suspension was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were 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 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/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. 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 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 (4% of cell viability when compared to the negative control) and variability (SD of % viability equal to 1.1). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤18 ), the assay was regarded as valid.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using one alive treated tissue without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using killed tissues and compared with negative control performed with alive tissues. Based on the results obtained (NSC=28.6% and NSMTT=18.5%), additional background subtractions were performed for the evaluation of irritant properties of the test item. The test item did not induce cell death in any replicate with a mean cell viability of 98.7% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 2.9 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item is classified as not irritaning to the skin.

Furthermore, in the acute dermal toxicity study in rats at the limit dose of 2000 mg/kg body weight, no adverse systemic or local effects were noted.

The potential of the test item to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 26 July 2013), the Guidance Document OECD series on testing and assessment no. 160 and the method described in the document issued by U.S. EPA/OPP (31 May 2013). The test item was spread on each epithelial surface of three idoneous bovine corneas as supplied (150 mg/cornea); 0.75 mL of physiological saline was then added on each cornea in order to better distribute the substance on the cornea surface (final concentration on cornea = 200 mg/mL, approximately 20% w/v); an exposure period of 4 hours was used. The mean opacity detected with an opacitometer at the end of the test item exposure period was 30.3. This was confirmed at the macroscopic observation in which slight opacity of the treated corneas was noted, although test item particles, adhering on the surface of all treated corneas, were observed. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 0.00. The calculated in vitro irritancy score (IVIS) for the test item was 30.3. Positive and negative controls [a 20 % (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in similar conditions and gave the expected results. According to the OECD Guideline no. 437, the test item can not be clearly classified.

The acute eye irritation of the substance was investigated in rabbits according to OECD 405.

A 0.1 g aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours after dosing.

No irritation at either the conjunctivae, iris or cornea (score of 0) was recorded in any treated animal, during the whole observation period of the study (72 hours). There were no signs of pain/distress after dosing.

There was no indication of a systemic effect. Blue staining of the fur around the treated eye, due to the colour of the test item, was noted in the animals during the whole observation period.

Changes in body weight were not remarkable.

These results indicate that the test item has no irritating effects on the eye of the rabbit.

Justification for classification or non-classification

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would suggest the following:

Classification: No category

Signal word: No signal word required

Hazard statement: No hazard statement required