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Administrative data

Description of key information

Skin irritation in vitro (OECT TG 439): not skin irritating

Eye irritation in vitro (OECT TG 437 / OECD TG 492): serious eye 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:
21 Sep 2017 - 16 Oct 2017
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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors 17-EKIN-041
Source strain:
other: 09-KERA-007 09-KERA-010
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): Batch no.: 17-EKIN-041
- Production date: 10 Oct 2017
- Date of initiation of testing: 10 Oct 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: untill all test item was removed
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
- Concentration (if solution): Undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25µL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main
Value:
101
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:
- OTHER EFFECTS:
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: refer to table

Historical Control Data for In Vitro Skin Irritation Studies

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Range

0.676 – 1.336

0.036 – 0.549

Mean

1.01

0.16

SD

0.16

0.10

n

155

154

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2013 to November 2016.

Interpretation of results:
other: not irritant
Remarks:
in accordance with Annex I of 1272/2008/EC (CLP)
Conclusions:
Based on the results of this study 3-methyl sulfolane does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

3-Methyl Sulfolane was evaluated for its ability to induce skin irritation on a human three dimensional epidermal model (OECD TG 439 (EPISKIN-SMTM)). The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 10% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 5%, indicating that the test system functioned properly.

In conclusion, 3-Methyl Sulfolane is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according tothe criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 Oct 2017 - 17 Oct 2017
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:
July 26, 2013
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals: 6-60 months
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline without antibioticsin a suitable container under cooled conditions.
- indication of any existing defects or lesions in ocular tissue samples: eyes exhibiting defects were discarded
- Indication of any antibiotics used: none used
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mL
Duration of treatment / exposure:
10 +/- 1 minutes
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes
Number of animals or in vitro replicates:
single sample, measured in triplicate
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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 32C. The corneas were incubated for the minimum of 1 hour at 32 C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Unexposed

POSITIVE CONTROL USED
Ethanol

APPLICATION DOSE AND EXPOSURE TIME
Undiluted 10 min exposure time

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 2

- POST-EXPOSURE INCUBATION: yes 120 +/- 10 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea using an opacitometer
- Corneal permeability: microtiter plate reader (OD490) TECAN Infinite® M200 Pro Plate Reader
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: according to guideline
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
IVIS > 3 ≤ 55, no prediction on the classification can be made according to UN GHS
Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Range of historical values if different from the ones specified in the test guideline: see table

Historical Control Data for the BCOP Studies

 

Negative control

Positive control

 

Opacity

Permeability

In vitroIrritancy Score

In vitroIrritancy Score

Range

-2.9 – 3.0

-0.016 – 0.042

-2.8 – 3.0

34.7 – 78.2

Mean

0.08

0.01

0.17

56.01

SD

1.04

0.01

1.14

12.51

n

84

77

78

55

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Aug 2014 to Aug 2017.

Interpretation of results:
other: Not determinable
Remarks:
in accordance with Annex I of 1272/2008/EC (CLP)
Conclusions:
Based on the results of the present study, 3-Methyl Sulfolane induced an IVIS > 3 ≤ 55. Therefore no prediction on the classification can be made according to UN GHS and Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The eye irritation hazard potential of 3-Methyl Sulfolane was as measured Acoording to OECDTG437 (BCOP test). The eye damage of the test substance was tested through topical application for 10 minutes.  The test item was applied as it is (750 µl) directly on top of the corneas. The negative and positive were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  3-Methyl Sulfolane induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 11 after 10 minutes of treatment.

In conclusion, since 3-Methyl Sulfolane induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.  

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 Nov 2017 - 17 Nov 2017
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:
09 October 2017
Deviations:
no
GLP compliance:
yes
Species:
human
Strain:
not specified
Remarks:
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q)
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability : In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
All cells used to produce EpiOcular™ are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions. In all cases, consent was obtained by these institutions from the donor or the donor's legal next of kin, for use of the cells or derivatives of the tissue for research purposes. The cells used to produce EpiOcular™ tissue are screened for potential biological contaminants. Tests performed for each of the potential biological contaminant listed in the analysis that follows, where performed according to the test method given. The product resulted in "no detection" for the following potential biological contaminants determined by the stated test method:
Keratinocytes:
HIV-1 virus - Oligonucleotide-directed amplification
Hepatitis B virus - Oligonucleotide- directed amplification
Hepatitis C virus - Oligonucleotide- directed amplification
Bacteria, yeast, and other fungi - long term antibiotic, antimycotic free culture
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl
- Concentration (if solution): undiluted
Duration of treatment / exposure:
30 ± 2 minutes at 37.0 ± 1.0°C
Duration of post- treatment incubation (in vitro):
12 ± 2 minutes
Number of animals or in vitro replicates:
tissues per test item
Details on study design:
- Details of the test procedure used :
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

- RhCE tissue construct used, including batch number :
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
3-Methyl Sulfolane exposure (30 ± 2 minutes at 37.0 ± 1.0°C), and 12 ± 2 minute (Post-Soak)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: both the MTT Color interference test and direct MTT reduction test indicated no interference.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Description of the method used to quantify MTT formazan : The amount of extracted formazan is determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) ifthe mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required. The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) ifthe mean percent tissue viability after exposure and post exposure incubation is less than or equal (:S) to 60%.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should be > 0.8 and <2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.
In case of color interference and/or MTT interacting test items:
a) The %NSCiiving should be~ 50% relative to the negative control OD.
b) The non-specific MTT reduction should be~ 50% relative to the negative control OD.

- Complete supporting information for the specific RhCE tissue construct used : available as certificate of analysis

- Positive and negative control means and acceptance ranges based on historical data : refer to table
Irritation parameter:
other: Tissue viability
Remarks:
(mean)
Run / experiment:
Main
Value:
5.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: refer to table

Historical control values

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.077 – 1.805

0.029 – 0.793

2.11 – 48.25

Mean

1.52

0.42

26.86

SD

0.21

0.23

14.11

n

16

16

16

SD = Standard deviation

n = Number of observations

 

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2013 to May 2017.

Interpretation of results:
other: Cat.1 eye irritant
Remarks:
in accordance with Annex I of 1272/2008/EC (CLP)
Conclusions:
Based on the results of this study 3-Methyl Sulfolane is potentially irritant or corrosive. The test item needs to be classified as a Category 1 or 2 eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The objective of this study was to evaluate the eye hazard potential of 3-Methyl Sulfolane, in a Reconstructed Human EpiOcular™ Model.The possible eye hazard potential of 3-Methyl Sulfolane was tested through topical application for 30 minutes. 3-Methyl Sulfolane was applied undiluted (50 µl) directly on top of the tissue for 30±2 minutes.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The results obtained with the positive and negative control were considered valid, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with 3-Methyl Sulfolane compared to the negative control tissues was 5%. Since the mean relative tissue viability for 3-Methyl Sulfolane was below 60% after 30±2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation in vitro (OECD TG 439)

3-Methyl Sulfolane was evaluated for its ability to induce skin irritation on a human three dimensional epidermal model (OECD TG 439 (EPISKIN-SMTM)). The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.

The positive control had a mean cell viability of 10% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 5%, indicating that the test system functioned properly.

In conclusion, 3-Methyl Sulfolane is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Eye irritation in vitro (OECD TG 437 & 492)

Two in vitro studies were available for this endpoint which were used as part of a weight of evidence approach:

In the first study, the eye irritation hazard potential of 3-Methyl Sulfolane was determined according to OECDTG437 (BCOP test). The test substance was tested through topical application for 10 minutes.  The test item was applied as it is (750 µl) directly on top of the corneas. The negative and positive control were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 3-Methyl Sulfolane induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 11 after 10 minutes of treatment. In conclusion, since 3 -Methyl Sulfolane induced an IVIS > 3 ≤ 55, meaning no prediction can be made if the substance can cause serious eye damage.

In the second study, the eye hazard potential of 3-Methyl Sulfolane was evaluated in a Reconstructed Human EpiOcular™ Model. The possible eye hazard potential of 3-Methyl Sulfolane was tested through topical application for 30 minutes. 3-Methyl Sulfolane was applied undiluted (50 µl) directly on top of the tissue for 30±2 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The results obtained with the positive and negative control were considered valid, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30±2 minutes treatment with 3-Methyl Sulfolane compared to the negative control tissues was 5%. Since the mean relative tissue viability for 3-Methyl Sulfolane was below 60% after 30±2 minutes treatment it is considered to be potentially irritant or corrosive to the eye.

From this available data it is not possible to conclude on a classification for 3 -methyl sulfolane. The result of the BCOP test does not conclude on serious eye damage, where the EpiOcular test indicates the substance should be at least regarded a serious eye irritant. In addition, the available data with regard to skin irritation (OECD 439 & 402) does not support the substance being seriously irritating as the result is negative in both tests. Taking this together with the eye irritation results, it is considered safe to conclude that 3-methyl sulfolane is a serious eye irritant and does not induce serious eye damage.

Justification for classification or non-classification

Based on the available information, 3-methyl sulfolane does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation.

Based on the available information, 3-methyl sulfolane is considered to be an eye irritant (Eye Irrit. 2 / H319) in accordance with the criteria outlined in Annex I of the CLP Regulation.