Registration Dossier

Administrative data

Description of key information

Skin Irritation / Corrosion

The potential of SOLVENT N1 to be irritating to the skin was investigated in the in vitro skin irritation test [reconstructed human epidermis model EPISKIN model] conducted according to the method described in

OECD guideline No. 439 and performed in compliance with good laboratory practices GLP.

SOLVENT N1 was found not-irritant and not-corrosive to the skin.

 

Eye Irritation / Corrosion

The potential of SOLVENT N1 to be irritating to the eye was investigated in the in vitro eye irritation test [reconstructed human cornea-like epithelium EpiOCular model] conducted according to the method described in

OECD guideline No. 492 and performed in compliance with good laboratory practices GLP.

SOLVENT N1 was found not-irritant and not-corrosive to the eye.

 

Respiratory Irritation

The potential of SOLVENT N1 to be irritating to the respiratory tract was assessed basing on the results of the acute

toxicity study conducted on rats. Effects on breathing functionality were observed during and immediately after exposure suggesting a possible irritant effects of SOLVENT N1 on the respiratory tract. However the severity of the observed effects is considered not sufficient for the application of the Hazard Class STOT SE 3 Respiratory Tract Irritation.

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:
16-MAR-2012 to 10-AUG-2012
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:
22 July 2010
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)
Specific details on test material used for the study:
- Lot/batch No.: SETTEMBRE 2011
- Expiration date of the lot/batch: 31-DEC-2016
- Storage condition of test material: ambient condition

- Purity = 74.152% w
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: N/A
Details on animal used as source of test system:
N/A
Justification for test system used:
The system is described in OECD TG No. 439
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM: Reconstructed human epidermal tissue (0.38 cm²).
- Source: SkinEthic Laboratories (France)
- Batch: 12-EKIN-024
- Examination before experiments: temperature indicator: pale grey (suitable for use); pH indicator: orange (suitable for use)

CULTURE
The test system was placed in a 12-well plate (supplied) in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium. Culture dishes were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

PRELIMINARY TEST
- Non-specific reduction of MTT was evaluated as follows:
Two mL of MTT Ready-to-use Solution was incubated with 10 microliters of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

- Colouring potential test:
Chemicals’ colouring potential was assessed for potential interaction with the test system.
10 microL of the test item was added to 90 microL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes.
Colouring of the solution at the end of the incubation time was evaluated.

MAIN ASSAY
A Main Assay was carried out including the test item, positive and negative controls ( 3 replicates, each).

Exposure period:
An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature.
Adding was carried out staggering samples of approximately 1 minute.
Positive control: an intermediate re-spreading was carried out approximately after 7 minutes of incubation.

Washing:
At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

Post-exposure period:
A 42 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.

Media collection for further analysis:
At the end of the incubation period, the plates were shaked on a plate shaker for about 15 minutes at approximately 300 rpm/min.
A volume of 1.6 mL of each incubation medium was removed and stored at -20°C for possible future analysis. No analysis was carried out on these samples, in agreement with the Sponsor.

MTT Assay:
The tissue insert and controls were incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity.
At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 microL of acidic isopropanol.
Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction.
At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 microL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 microL) of acidic isopropanol were analysed and used as blank.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Number of replicates:
3
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied, concentration: 20 µL/epidermis unit each measuring 0.38 cm² (treatment level: 53 µL/cm²)
Duration of treatment / exposure:
15 ± 0.5-min exposure period
Observation period:
Following the 15-min exposure period, the tissues were washed with phosphate buffered saline (PBS). Then all tissues were incubated in SkinEthic maintenance medium for 42 hrs at 37°C, 5% CO2 and saturated humidity.
Number of animals:
None: in vitro method
Details on study design:
REMOVAL OF TEST MATERIAL
- Washing: at the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.
- Time after start of exposure: 15 min

REMARKS: the test item was applied as supplied in three replicates. Tissues were exposed to the test item in a ventilated cabinet at room temperature. Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate (SDS) solution in water and Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit.

SCORING SYSTEM: the tissue insert and controls were incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hrs at 37°C, 5% CO2 and saturated humidity. At the end of the 42-hr incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions. The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were mixed by vortexing and preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank.

ACCEPTABILITY CRITERIA
Acceptability criteria were determined following set-up and validation studies.
- EPISKIN™ batch acceptance:
* Negative controls: OD values of samples ≥ 0.600, CV% ≤ 18.
* Positive controls: mean viability expressed as percentage of the negative control ≤ 40% and CV% ≤ 18.
- Test item data acceptance: CV% ≤ 18.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main test
Value:
92.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
CV% = 5.5

Table 3: Results of the main test

 

Optical density

(OD)595

Mean ±

standard deviation (SD)

CV%

OD-blank

Mean ±

standard deviation (SD)

CV%

% of viability*

Blank

0.048

0.054

0.053

0.054

0.049

0.047

0.051 ± 0.003

5.9

-

-

-

-

Negative control

0.831

0.820

0.868

0.920

0.829

0.882

0.858 ± 0.039

4.6

0.780

0.769

0.817

0.869

0.778

0.831

0.807 ± 0.039

4.8

100%

Positive control

0.085

0.065

0.070

0.064

0.084

0.074

0.074 ± 0.009

12.2

0.034

0.014

0.019

0.013

0.033

0.023

0.023 ± 0.009

39.1

2.9

Test item

0.739

0.767

0.817

0.852

0.832

0.768

0.796 ± 0.044

5.5

0.688

0.716

0.766

0.801

0.781

0.717

0.745 ± 0.044

5.9

92.3

*: referred to the negative control mean value.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the established criteria for positive results (cell viability less than 50%), the test item is considered to have no irritant effect on the skin under the reported experimental conditions.
Executive summary:

The potential of the test item SOLVENT N1 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKINTM. The study was conducted according to OECD guideline 439 and in compliance with good laboratory practices (GLP).

 

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 hour recovery period. The final endpoint of the assay was the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability.

 

Prior to the main assay, a preliminary test was carried out to assay the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and of colouring water per se.

No interaction was recorded between the test item and MTT in test conditions similar to those of the main assay. Moreover, no colouring potential of the test item in contact with water was recorded. Thus, no additional control was added in the main phase for the evaluation of non-specific coloration which may influence evaluation of results.

 

In the main assay, the test item was applied as supplied in three replicates at the treatment level of 20 µL/epidermis unit each measuring 0.38 cm² (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)] 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 and variability, in agreement with the guideline indications. According to the method, the mean value was considered the baseline value of the experiment and thus represented 100% of cell viability.

The positive control caused the expected cell death (2.9% of cell viability when compared to the negative control) with the expected variability (CV% = 12.2).

Therefore, the assay was regarded as valid.

The test item did not induce cell death, with a mean cell viability of 92.3% when compared to the negative control. Good homogeneity was observed between replicates (CV% = 5.5).

The test item result was therefore accepted as valid.

 

According to the established criteria for positive results (cell viability less than 50%), the test item was considered to have no irritant effect on the skin under the reported experimental conditions. Therefore, the test material does not meet the classification criteria of EC Regulation No. 1272/2008 (CLP / EU GHS) and UN GHS for skin irritation.

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:
August to November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 24/02/2016
- Expiration date of the batch: July 2021
- Purity test date: 75.6 % (w/v) N1 SOLVENT

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of the test substance: the substance is chemically stable at room temperature
- Storae conditions: In the refrigerator set at +5°C (for reducing the risk of evaporation)
- Correction factor: none
Species:
human
Strain:
other: not applicable.
Remarks:
Reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
Description: the EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The cells are cultured in proprietary serum-free culture media, which induces corneal differentiation and the formation of the organotypic 3D cornea-like model. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.
Quality Control: the acceptability range for the barrier function as measured by the ET50 assay is established by the RhCE tissue construct developer/supplier to be between 12.2 and 37.5 min. Data demonstrating compliance with all production release criteria were provided by the RhCE tissue construct supplier and the information are documented in the certificate of analysis archived in the study files.
Selection: at receipt, the tissues were inspected for obvious defects as they could have been rejected based on blistering, excess fluid or air bubbles below the tissue insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
Expiry date: the EpiOcular tissues were used within 72 hours of their production.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Undiluted test item was applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues.
Duration of treatment / exposure:
Pre-incubation: 1 hour (+37°C, 5% CO2 in a humidified incubator). After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
Exposure: The tissues were incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.
Number of animals or in vitro replicates:
Main test:
One 6-well plate was used for the test item-treated tissues.
Positive and negative controls were placed on separate 6-well plates (one plate for each).
Test item, negative and positive controls were applied on duplicate tissues.
Details on study design:
PRELIMINARY TESTS

1) Test for direct MTT reduction with the test item:
As a test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test. This property of the test item would only be a problem if, at the time of the MTT viability test (after rinsing), there remained a sufficient amount of test item present on or in the tissue. In this case, the true metabolic MTT reduction and the false direct MTT reduction should be differentiated and quantified.
To identify any test substance interference with the MTT endpoint, the following preliminary test was performed:
. 50 μL of the test item were added to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
. a negative control was tested concurrently by adding 50 μL of sterile deionized water to 1 mL of a 1.0 mg/mL freshly prepared MTT solution,
. both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the color of the solutions obtained was evaluated.
If the MTT solution color turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT.

2) Test for the detection of the coloring potential of the test item:
As a test item may be colored or become colored in contact with water and/or isopropanol, it is necessary to test its potential interference with the MTT determination in these two conditions.
The maximum amount of test item, 50 μL was added to 1 mL of water and incubated for at least 1 hour in the dark at +37°C, 5% CO2 and 2 mL of isopropanol, incubated in a 6-well plate and placed on an orbital plate shaker for 2 hours at room temperature. After that, the presence and intensity of the coloration were evaluated. If the solution changes color significantly, additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.

MAIN TEST:

Pre-incubation of the tissues:
As the tissues were shipped before the day prior the treatment, tissues were stored between +2°C and +8°C, prior their pre-incubation. The tissues were equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. The underside of each tissue was inspected for air bubbles between the agarose gel and the insert. Tissues with air bubbles under the insert covering greater than 50% of the insert area were not used. Any unusual observation was noted separately. The plastic bag containing the 24-well plate shipping container was disinfected by wiping with 70% ethanol-soaked tissue paper. Each 24-well shipping container was then removed from its plastic bag under sterile conditions. A volume of 1 mL assay medium pre-warmed (+37°C) was added to 2 wells per pre-labeled 6-well plate. The tissues were carefully removed from the 24‐well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze.
The insert was then transferred aseptically into the 6-well plate and pre-incubated at +37°C, 5% CO2 in a humidified incubator for 1 hour. After the pre-incubation period, the assay medium was removed and replaced by 1 mL of fresh assay medium before incubation overnight (16-24h) at +37°C, 5% CO2 in a humidified incubator.
Each 6-well plate was labeled with the test item or control codes.

Treatment of tissues:
Following the pre-incubation period, the tissues were pre-wetted with 20 μL of D-PBS. Each insert was tapped on the walI of the plate to ensure that the entire tissue surface was wetted with D-PBS. The tissues were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 (± 2) minutes.
The test item, negative and positive controls were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were then tapped on the wall of the plate to ensure that the items were applied evenly to the surface of each tissue.
All treated plates (including those treated with positive and negative controls) were covered by a plate sealer in order to avoid any evaporation/volatility of test item.
Then, all tissues (test item, negative and positive controls) were incubated at +37°C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).
The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue receives an equal exposure period.

Rinsing of tissues:
At the end of the treatment period, each tissue was removed from the well of the treatment plate and rinsed to gently remove any residual test or control items. A set of three clean beakers containing a minimum of 100 mL each of D-PBS was used per test or control item. The tissues were rinsed two at time by holding replicate inserts together. The test or control items were firstly decanted from the tissue surface onto a clean absorbent paper. The tissues were then dipped into the first beaker of D-PBS, swirled in a circular motion during approximately 2 seconds, lifted out and decanted back into the beaker. This process was performed three times per beaker. Any remaining liquid was decanted onto an absorbent paper.

Post-soak and post-incubation:
The rinsed tissues were transferred to new wells of a pre-labeled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature. This incubation in assay medium was intended to remove any test article from the tissue.
Each tissue was incubated for 12 minutes (± 2 minutes) at room temperature to remove any liquid test item or negative and positive controls from the tissue.
At the end of the Post-soak immersion, each insert was blotted on absorbent material and transferred to appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium. The tissues were then incubated for 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator for test item, negative and positive controls.

MTT viability assay:
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution (1.0 mg/mL) was added into new wells of pre-labeled 24-well plates.
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were then transferred to the MTT pre-filled wells and incubated for 3 hours (± 10 minutes) at +37°C, 5% CO2 in a humidified incubator.
At the end of the 3-hour incubation period, the underside of each tissue was blotted on absorbent paper to dry. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated.
For the test item, negative and positive control-treated tissues, the inserts were transferred to new wells of the 24-well plate containing 2 mL of isopropanol per well so that isopropanol was flowing into the insert on the tissue surface.
Plates were surrounded with parafilm to prevent evaporation. Formazan extraction was performed overnight at +2-8°C and protected from light.

Optical Density measurements:
At the end of the formazan extraction period, tissues (test item, negative and positive control-treated tissues) were pierced.
The extract solution was mixed using a pipette and two 200 μL aliquots were transferred to the appropriate wells of a pre-labeled 96-well plate.
One 96-well plate was used for the negative and positive controls (placed at opposite end of the plate) and a separate 96-well plate was used for test item-treated tissues.
For each 96-well plate, the average Optical Density value (OD) of 4 wells containing 200 μL of isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.

Data analysis:
As the test item was found in the preliminary test not to have any coloring potential and any direct MTT reducing properties, no additional controls were run during the main test. Therefore, the mean blank OD value (mean ODblank) was calculated from the four replicates.
Then, the mean ODblank was subtracted from each OD value and the corrected mean OD values (mean cOD) of the two aliquots were calculated for each tissue.
The corrected mean OD of the two negative controls-treated tissues (mean cODNC) was set to 100% viability and was used as a reference.
For the tissues treated with the test item or negative or positive controls, the relative viabilities for each tissue were expressed as percentages of the reference viability and were calculated as follows:
TI relative viability (%) = (cOD TI or NC or PC / mean cOD NC) x 100
with:
cOD TI = corrected OD of each tissue treated with test item.
cOD NC = corrected OD of each tissue treated with negative control.
cOD PC = corrected OD of each tissue treated with positive control.

Acceptance criteria
The results of the study were considered acceptable if the following criteria are fully met:
. the mean cOD of the negative controls is between 0.8 and 2.5,
. relative mean viability of the positive control is < 50% of the relative mean viability of the negative control,
. the difference of viability between the two tissue replicate is < 20%.
Irritation parameter:
other: relative mean viability (%)
Run / experiment:
Main test / Mean value of the two treated tissues. Difference of 2% between duplicate tissues.
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: FULFILLED.
Mean cOD of the negative control = 1.763 (SD = 0.066). (Acceptance criteria: Mean cOD of the negative control between 0.8 and 2.5)

- Acceptance criteria met for positive control: FULFILLED.
Relative mean viability of the positive control = 26% (SD = 5%). (Acceptance criteria: Relative mean viability of the positive control < 50% of the relative mean viability of the negative control)

. Acceptance criteria: difference of viability between the two tissue replicate is < 20%. FULFILLED
Difference of viability between the two tissue replicate:
2% for test item treated tissues
5% for negative control tissues
7% for positive control tissues
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, N1 SOLVENT, is considered to be non-irritant to Reconstructed human Cornea-like Epithelium.
According to the results of this study, the classification of the test item should be: "No Category" (GHS 2015 and Regulation (EC) No. 1272/2008).
Executive summary:

The study was conducted according to OECD Guideline No. 492 and in compliance with the principles of Good Laboratory Practice. No deviation from OECD Guideline No. 492 occurred.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its coloring potential.

Following the preliminary tests, the eye irritation potential of the test item was assessed in the main test. The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 30 minutes.

As the test item is a volatile liquid, measures for avoiding losses due to evaporation and ensuring appropriate exposure of tissues were applied during the test. Indeed, all treated plates (including those treated with positive and negative controls) were covered by a plate sealer in order to avoid any evaporation/volatility.

At the end of the treatment period, each tissue was rinsed with D-PBS, incubated for 12 minutes at room temperature, blotted on absorbent material, and then incubated for another 2 hours at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Mean viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

In the main test, all the acceptance criteria were fulfilled. The study is therefore considered valid.

The relative mean viability of the tissues treated with the test item was 99% with a difference of 2% between duplicate tissues. As the mean viability was > 60% after the MTT reduction, the results met the criteria for a non-irritant response.

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

Respiratory irritation

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

Additional information

Skin Irritation / Corrosion

The potential of SOLVENT N1 to be irritating to the skin was investigated in a study conducted according to OECD guideline 439, Reconstructed Human Epidermis Test Method and in compliance with good laboratory practices (GLP).

The in vitro method described in the OECD guideline 439 allows to discriminates skin irritants (Cat. 2) from chemicals not classified for skin irritation (No Cat.) according to the EU GHS classification system. Irritant chemicals are identified by their ability to decrease tissue viability below 50% of the negative control.

According to OECD No. 439 method, a result indicating skin irritation (Cat. 2) does not allow excluding corrosion (Cat. 1) therefore in case the test showed a tissue viability below 50% of the negative control for the test chemical, an in vitro skin corrosion test would be required to determine the final classification (Cat. 2 (irritant) or Cat. 1(A, B or C) (corrosive)).This was not the case for the tissues treated with SOLVENT N1 which showed a mean tissue viability = 92.3% compared to the negative control, therefore no further test is necessary and it can be concluded that the substance does not require classification and labelling according to EU GHS Criteria.

 

Eye Irritation / Corrosion

The potential of SOLVENT N1 to be irritating to the eye was investigated in an in vitro study conducted according to OECD Guideline 492, Reconstructed Human Cornea-like Epithelium (RhCE) Test Method and in compliance with good laboratory practices (GLP).

The method described in the OECD guideline 492 allows toIdentify Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage according to EU GHS and UN GHS classification system.

According to OECD Guideline 492 method, chemicals not requiring classification and labelling according to UN GHS are identified as those having a tissue viability higher than (>) 60% compard to the one of the negative control.

The method is not intended to differentiate between UN GHS Cat. 1 (serious eye damage) and UN GHS Cat. 2 (eye irritation), so in case the viability of the tissues treated with the test item was below than (<) 60% further evaluation would be needed. This was not the case for the tissues treated with SOLVENT N1 which showed a mean tissue viability = 99% compared to the negative control, therefore no further test is necessary and it can be concluded that SOLVENT N1 does not require classification and labelling according to EU GHS Criteria.

 

Respiratory Irritation

The potential of SOLVENT N1 to be irritating to the respiratory tract was assessed basing on the results of the acute toxicity study conducted on rats. The study was conducted according to OECD guideline 403 and in compliance with good laboratory practices (GLP). A group of five male and five female rats was exposed for a single 4-hour period to vapours of SOLVENT N1 at a chemically determined mean atmosphere concentration of 5080 ± 213 ppm SOLVENT N1 / air (corresponding to ca. 54 mg/L). There were no deaths or other indications of severe toxicity during the study but some signs suggesting a possible irritant effects of SOLVENT N1 on the respiratory tract were observed during and immediately after exposure: decreased breathing depth and increased breathing rate were observed during exposure in all animals, and salivation in two animals and abnormal respiration in one female was observed immediately after exposure. There were no clinical changes during the remaining 14 day-observation period.

Considering that the test was conducted at the limit concentration of ca. 50 mg/L which is more than twice the limit concentration recommended in the current standard guidelines for assessing acute toxicity and that the observed effects on the respiratory tract were reversed in 90% of the animals immediately after exposure, the observed effects are considered not relevant for the application of the Hazard Class STOT SE 3 Respiratory Tract Irritation.

Justification for classification or non-classification

Skin Irritation / Corrosion

The in vitro study described in the OECD guideline 439 allows to discriminates skin irritants (Cat. 2) from chemicals not classified for skin irritation (No Cat.) in member countries or regions that do not adopt the optional UN GHS Category 3 (mild irritants), like Europe.

Irritant chemicals are identified by their ability to decrease tissue viability below 50% of the negative control.

According to the results, SOLVENT N1 was found not-irritant to the skin (mean tissue viability = 92.3% compared to the negative control) therefore not requiring classification and labelling according toRegulation (EC) No. 1272/2008 (EU GHS).

 

Serious Eye Damage / Eye Irritation

The in vitro study described in the OECD guideline 492 allows to Identify Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage according to EU GHS and UN GHS classification system.

According to OECD Guideline 492 method, chemicals not requiring classification and labelling according to UN GHS are identified as those having a tissue viability higher than (>) 60% compard to the one of the negative control. According to the results, SOLVENT N1 was found not requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (mean tissue viability = 99% compared to the negative control) according to Regulation (EC) No. 1272/2008 (EU GHS).

 

Respiratory Irritation

Effects on breathing functionality were observed in rats during and immediately after exposure to high concentration of SOLVENT N1 vapours in air.

The reported effects were observed in a study that was conducted at the limit concentration of ca. 50 mg/L which is more than twice the limit concentration recommended in the current standard guidelines for assessing acute toxicity on animals. Moreover the observed effects were reversed in 90% of the animals immediately after exposure. The observed effects are therefore considered not relevant for the application of the Hazard Class STOT SE 3 Respiratory Tract Irritation.