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Description of key information

In vitro studies for skin and eye irritation were conducted.

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:
From 2013-09-26 to 2013-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD guidelines and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The 12 wells plates with tissues were preincubated for 2h instead of 24h. Upon receiving instructions from the supplier, Episkin tissues were transferred to maintenance medium for 24h. This protocol deviation did not affected the study integrity.
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
yes
Remarks:
The 12 wells plates with tissues were preincubated for 2h instead of 24h. Upon receiving instructions from the supplier, Episkin tissues were transferred to maintenance medium for 24h. This protocol deviation did not affected the study integrity.
GLP compliance:
yes
Species:
other: EPISKIN Small Model (human)
Details on test animals or test system and environmental conditions:
ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment.
-Humid atmosphere of 80 - 100% (actual range 82 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark
-Temperature:37.0 ± 1.0°C (actual range 36.3 – 37.6°C).
Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Type of coverage:
open
Vehicle:
physiological saline
Amount / concentration applied:
10.0 to 13.3mg
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SYSTEM
Name: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-034)
Source: SkinEthic Laboratories, Lyon, France.
Rationale: One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.0 to 13.3 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
% tissue viability
Value:
112
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
no indication of irritation


Table 1 Mean absorption in thein vitroskin irritation test with the test substance

A

 B 

 

C

  Mean(OD570) +/-SD Negative Control 1.180

1.134 1.050  1.121 +/- 0.066

  The test substance 1.2021.2841.286  1.257 +/- 0.048

 


Table 2 Mean tissue viability in thein vitroskin irritation test with the test substance

 Mean tissue viability (Percentage od control)  Negative Control 100 The test substance 101 Positive Control 6

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other:
Conclusions:
The test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
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:
From 2013-09-26 to 2013-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD guidelines and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes
Species:
other: Bovine eyes
Details on test animals or tissues and environmental conditions:
Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 ± 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)).
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws.
The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
326 to 391 mg (A4N3 was applied directly on the corneas in such a way that the cornea was completely covered).
Duration of treatment / exposure:
240+/-10 minutes
Number of animals or in vitro replicates:
Three eyes for the test substance, negative control and positive control substance.
Details on study design:
Treatment of corneas and opacity measurement
The medium from the anterior compartment was removed and 750 μl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. A4N3 was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (326 to 391 mg).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Permeability determination
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
0.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
-0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
-2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The individual in vitro irritancy scores for the negative controls was 0.0 for all three corneas. The individual positive control in vitro irritancy scores ranged from 103 to 133. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with A4N3 showed opacity values ranging from -2 to 0 and permeability values ranging from -0.005 to 0.022. The corneas were clear after the 240 minutes of treatment with A4N3. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.0 to 0.3 after 240 minutes of treatment with A4N3.

Summary of opacity, permeability and in vitro scores

 Treatment Mean Opacity Mean Permeability Mean In vitro Irritation Score Negative control 00.000  0.0 Positive control 1001.400  121 The test substance -10.005-0.9 

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Since the test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

No indication of irritation were observed.


Justification for selection of skin irritation / corrosion endpoint:
Only study available

Justification for selection of eye irritation endpoint:
Only study available

Justification for classification or non-classification

The test substance was negative for irritation potential in the skin and eye in vitro screening test conducted and is therefore not classified as an irritant.