Registration Dossier

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Administrative data

Description of key information

A key dermal irritation study on the potential of 2-methylpropane-2-thiol to cause dermal irritation was evaluated. Following 4 hours occlusive exposure, the test material was reported to not cause skin irritation (Latven, 1976).

In an in vitro bovine corneal opacity and permeability study with the registered substance 2-methylpropane-2-thiol, conducted according to OECD Test Guideline 437 and in compliance with GLP, the average IVIS score was 17, which does not allow a prediction of potential for corrosion to the eye (Charles River Laboratories, 2020a).

In an in vitro eye irritation test on EpiOcular™ reconstructed human cornea-like epithelium with the registered substance 2-methylpropane-2-thiol, conducted according to OECD Test Guideline 492 and in compliance with GLP, 3% cell viability was obtained which gives a positive indication of irritation (Charles River Laboratories, 2020a).

In an in vivo eye irritation study for the structural analogue, propane-1-thiol (NPM), conducted according to a protocol similar to OECD Test Guideline 405 and in compliance with GLP, the registered substance was concluded to be not irritating to eyes (Moon, 1981).

In an in vivo eye irritation study for the structural analogue, iso-propyl mercaptan (IPM), conducted according to a national standard method with acceptable deviations, but prior to GLP, the registered substance was concluded to be not irritating to eyes (Latven, 1977).

No key data examining respiratory irritation was available for 2-methylpropane-2-thiol; however acute inhalation data from studies in rats and mice indicates that 2-methylpropane-2-thiol is not a respiratory irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
This study was classified as reliable with restrictions because the test procedure was in acc ordance with national standard methods with acceptable restrictions.
Qualifier:
according to guideline
Guideline:
other: US DoT guideline no. 49 CFR 173.240
GLP compliance:
no
Species:
rabbit
Strain:
other: Albino
Details on test animals or test system and environmental conditions:
no data
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
6
Irritation parameter:
erythema score
Basis:
animal: all
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal: all
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No irritant effects of any kind were discernible at any of the treated skin sites.
Interpretation of results:
GHS criteria not met
Conclusions:
In a skin irritation and corrosion study, 2-methylpropane-2-thiol was found to be not irritating.
Executive summary:

In a skin irritiation and corrosion study (49 CFR 173.240), six albino rabbits (sex and strain unspecified) received 0.5mL of undiluted 2 -methylpropane-2-thiol (CAS # 75 -66 -1) applied to the intact skin site under standard gauze patches. The entire fur-clipped trunk of each animal was then covered with an impervious sleeve for a skin-contact period of 4 hours and then observed during 3 days. No signs of

irritation were discernible at any of the six treated skin sites. The substance was classified as not irritating based on EC Regulation No 1272.

This study received a Klimisch score of 2 and was classified as reliable with restrictions because the test procedure was in accordance with national standard methods with acceptable restrictions.

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:
22 October 2019 - 23 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: bovine cattle (Bos taurus)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
- Transport from Supplier to Charles River Laboratories Evreux: the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Charles River Laboratories Evreux. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].


Upon arrival at Charles River Laboratories Evreux, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

- Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.
- Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
The corneas were then used immediately.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL per cornea
- Concentration: undiluted
Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds), following by rinsing
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes)
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item, negative control, positive control)
Details on study design:
EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any eyes with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incubation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
The corneas from the same series were always processed in the same order at each step. The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes in a water bath at +32°C (± 1°C).

RINSING OF THE CORNEAS
The purpose of rinsing was to eliminate as much item as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item and both negative and positive controls were removed from the front opening of the anterior chamber (open-chamber method) and the corneas were rinsed with pre-warmed cMEM containing phenol red. Rinsing of the test item-treated corneas was performed as follows:
. the anterior chamber was emptied using a gavage tube attached to a vacuum pump,
. the corneas were rinsed three to four times with pre-warmed cMEM containing phenol red (i.e. three times for both negative and positive controls-treated series and four times for the corneas treated with test item), until the test item had been completely removed from the chamber or until the phenol red
was not discoloured.
. then, all corneas were finally rinsed with pre-warmed cMEM without phenol red.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea.
The average change in opacity for the corneas treated with the negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with the test item or the positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas
treated with the negative control was negative, it is considered equal to 0. The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution (4mg/mL) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube.
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated with the test item or the positive control was calculated by subtracting the average negative control cornea OD490nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

The IVIS was calculated for each test item and positive control-treated cornea.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
17
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS (macroscopic examination):
Fluorescein fixation was observed on one out of the three corneas treated with the negative control.
Opacity and fluorescein fixation were observed on the three corneas treated with the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
. the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
. the mean opacity and mean OD490 nm of the negative control corneas should be less than the established upper limit of historical mean.
Interpretation of results:
other: As the mean IVIS was >3 and ≤55, the eye hazard potential of the test item could not be predicted.
Conclusions:
In an in vitro bovine corneal opacity and permeability test study, conducted according to OECD 437 and in compliance with GLP, the potential of the test material to be ocular corrosive or severe irritant could not be established.
The average IVIS score was 17 which is between >3 and ≤55 which according to the guideline means the eye hazard potential of the test item could not be predicted. To be able to confirm classification, further testing was recommended.
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 January 2020 - 23 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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:
14 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: reconstructed human Cornea-like epithelium (tissues)
Details on test animals or tissues and environmental conditions:
Cell type: non-transformed keratinocytes
Cell supplier: MatTek, Bratislava, Slovak Republic

Details on test system and justification for its use:
The EpiOcular™ model consists of an airlifted, living, multi-layered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a nonkeratinized 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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Concentration: neat (as supplied),
- Amount applied: 50 µL per tissue, taking care to spread it over the whole tissue surface area without damaging the tissue samples.
Duration of treatment / exposure:
Exposure period of 30 minutes (± 2 minutes) at +37°C, 5% CO2 in a humidified incubator, followed by rinsing.
Duration of post- treatment incubation (in vitro):
Post-soak period of 12 minutes (± 2 minutes) at room temperature.
Post-incubation period of 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.
Number of animals or in vitro replicates:
The test item and both negative and positive controls were applied on duplicate tissues.
At the end of the viability assay, formazan level in tissues was assessed in duplicate for each tissue.
The mean OD values were then calculated from the four replicates of each item.
Details on study design:
PRELIMINARY TESTS
* Test for Direct MTT Reduction with the Test Item
As the test item may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test its ability to directly reduce MTT before performing the main test. 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,
- a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 10 minutes).
Then the colour of both solutions obtained was evaluated. If the MTT solution colour turns blue/purple when compared to the negative control, the test item was presumed to reduce MTT and additional controls were performed on freeze-dead tissues in parallel to the main test, to evaluate the part of OD due to the non-specific reduction of the MTT. Otherwise, no additional tissue controls were used.
 
* Test for the Detection of the Colouring Potential of the Test Item
As a test item may be coloured or become coloured in contact with water or isopropanol, it is necessary to test its potential interference with the MTT determination.
The ability of the test item to absorb significantly light at the wavelength used for MTT determination was tested as follows: a volume of 50 µL of the test item was added to 1 mL of water and a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Plate was then incubated for at least 1 hour at +37°C, 5% CO2.
Then, two 200 µL aliquots of test item solution and water (blank) were transferred to a 96-well plate and absorbance was measured.
If, after subtraction of the blank OD, the OD of the test item solution was > 0.08 (approximately 5% of the mean viability of the negative control) the test item was considered as having colouring potential and additional controls were performed on viable tissues in parallel to the main test. Otherwise, no additional tissue controls were used.

MAIN TEST
One 6-well plate was used for each item: one for tissues treated with the test item, one for tissues treated with the positive control and another one for those treated with the negative control. Each item was applied on duplicate tissues.

In addition, as the test item was found to have direct MTT reducing properties in the preliminary test, additional controls (i.e. two freeze-dead tissues treated with the test item and two freeze dead tissues treated with the negative control) were added in the main test for the evaluation of the OD part due to non specific MTT reduction. One 6-well plate was used for each of these control types.
Freeze-dead tissues have no metabolic activity but absorb and bind the test item in the same way as viable tissues. It should be noted that freeze-dead tissues show a small amount of MTT reduction due to residual reducing enzymes within the tissue.
Freeze-dead tissues were prepared by placing untreated EpiOcular™ tissues in a 24 well plate. The tissues were then placed in a freezer (-20°C) overnight, thawing to room temperature, and then refreezing. Once frozen, the tissues were stored indefinitely in the freezer.

PRE-INCUBATION OF THE TISSUES
As the tissues were shipped the day prior the treatment, tissues were stored between +2°C and +8°C, prior their pre-incubation. The tissues were then equilibrated (in the 24-well shipping container) to room temperature for at least 15 minutes. Each tissue was inspected as specified in internal procedure, removed from the 24-well shipping containers, placed in a 6-well plate containing 1 mL of pre-warmed assay medium and then incubated for 1 hour (± 5 minutes) at +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. Each 6-well plate was labelled with the test item or control codes.

TREATMENT
Following the pre-incubation period, the tissues were pre-wetted with 20 µL of D-PBS, inserts were tapped to ensure that the entire tissue surface was wetted and plates were then incubated at +37°C, 5% CO2 in a humidified incubator for 30 minutes (± 2 minutes).
The test item, the negative control and the positive control were applied topically on each designated tissue, and gently spread onto the epithelium surfaces to ensure uniform covering of the tissues. Inserts were tapped on the wall/well of the plate to ensure that each item was correctly spread across the entire surface of each tissue.
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 (pre-wetting, treatment and rinsing) in the same order and at regular time intervals to ensure each tissue receives an equal exposure period. In addition, during pre wetting and treatment, a sealer was placed on each plate before putting the plastic lids back and incubating in the incubator, to avoid evaporation and/or cross-contamination.

RINSING
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. Items were first removed from the tissue surface by tapping upside down each insert onto a clean absorbent paper. 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-labelled 12-well plate containing 5 mL of assay medium pre-warmed at room temperature and were then incubated for 12 minutes (± 2 minutes) at room temperature, in order to remove any items 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-labelled 6-well plate containing 1 mL of assay medium. A sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Then, tissues were incubated for 2 hours (± 15 minutes) at +37°C, 5% CO2 in a humidified incubator.

MTT VIABILITY ASSAY
Following the post-treatment incubation, a volume of 0.3 mL of a freshly prepared MTT solution at 1.0 mg/mL was added into new wells of pre-labelled 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. Tissues were then transferred to the MTT pre-filled wells, a sealer was placed on each plate before putting the plastic lids back, to avoid evaporation and/or cross-contamination. Then, tissues were 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.
As the test item was a non-colourant liquid, each insert of tissues was 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, the plates were placed under orbital shaking at room temperature for 15 minutes prior using them. Then, all tissues (treated with the test item, the negative control and the positive control) were pierced.
The extract solution was mixed using a pipette and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 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 all test item-treated tissues (including additional freeze-dead). As the test item was found to have MTT reducing properties in the preliminary assay, another 96-well plate was also used for the negative control-treated freeze-dead tissues.
For each 96-well plate, the average Optical Density value (OD) of four wells containing 200 µL of isopropanol only was used as the blank. The OD was measured at 570 nm using a plate reader.

DATA ANALYIS
On each plate, 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 mean cOD of the two negative control-treated tissues (mean cODNC) was set to 100% viability and was used as reference.
For the tissues treated with the test item and the positive control, the relative viability of each tissue was expressed as percentage of the reference viability and were calculated as follows:
Relative viability (%) = (cODTI or PC / mean cODNC) x 100

With:
cODTI = corrected OD of each tissue treated with the test item,
cODNC = corrected OD of each tissue treated with the negative control,
cODPC = corrected OD of each tissue treated with the positive control.

As the test item was found to have direct MTT reducing properties in the preliminary test, two freeze-dead tissues treated with the test item and two freeze-dead tissues treated with the negative control were also run as additional controls in the main test (for evaluation of the non-specific MTT reduction - NSMTT-). For each of these tissues, the NSMTT was calculated as follows:
NSMTT (%) = [(cODTIdead - mean cODNC_dead) / mean cODNC] x 100

With:
cODNC_dead = corrected OD of each freeze-dead tissue treated with the negative control,
cODTIdead = corrected OD of each freeze-dead tissue treated with the test item.

The mean NSMTT (%) was calculated and was subtracted to the mean relative viability of the test item-treated tissues to obtain the True MTT metabolic conversion, as follows:
True MTT metabolic conversion (%) = mean TI relative viability (%) - mean NSMTT (%)

The difference of viability between two tissue replicates was also calculated for each item.
Irritation parameter:
other: % cell viability
Run / experiment:
mean
Value:
3
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
26%
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE CRITERIA
- the mean OD blank of each plate (i.e. extraction solvent) should be < 0.1,
- the mean cOD of the negative controls should be between 0.8 and 2.8,
- relative mean viability of the positive control should be < 50% of the relative mean viability of the negative control,
- the difference of viability between two tissue replicates should be < 20%.

EVALUATION OF THE COLOURATION OF TISSUES AT THE END OF THE MTT INCUBATION
The qualitative evaluation of the MTT staining was performed with the naked eye.
Viable test item-treated tissues appeared blue/white (i.e. white tissues with blue surrounds) which was considered to be indicative of semi-viable tissues.

EVALUATION OF MTT RESULTS
The relative mean viability of the tissues treated with the test item, corrected by the NSMTT, was 3% with a difference of 1% between duplicate tissues.
As the relative mean viability was < 60%, the acute eye irritation potential of the test item could not be predicted.
Interpretation of results:
other: further information required to distinguish between Category 1 and Category 2
Conclusions:
In an eye irritation test on EpiOcular™ reconstructed human cornea-like epithelium, conducted accord ing to OECD 492 and in compliance with GLP, 3% cell viability was obtained. This result gives a positive indication of irritation. The study cannot distinguish between Category 1 and 2 of Regulation (EC) No. 1272/2008.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980-12-19 to 1981-01-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The study is classified as reliable with restrictions because it complies with GLP and adheres to OECD guideline 405.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
Method: other: UBTL Protocol No. 01A-50
Similar to OECD 405 and OPPTS 870.2400
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: University of Utah vivarium or other reputable breeder
- Housing: housed individually in wire cages with no sawdust r other dust producing material in the room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70 +/- 3
- Humidity (%): 30 to 60
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hr / 12 hr
Controls:
yes, concurrent no treatment
Amount / concentration applied:
Amount applied: 0.1 ml
Duration of treatment / exposure:
4 seconds
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
6
Details on study design:
Comment: not rinsed
SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: hand-held ophthalmoscope and then with fluorescein
Irritation parameter:
overall irritation score
Remarks:
Draize system
Basis:
mean
Time point:
other: 1 hr.
Score:
8.3
Max. score:
14
Reversibility:
fully reversible
Irritation parameter:
overall irritation score
Remarks:
Mean Draize score
Basis:
mean
Time point:
other: 24 hrs.
Score:
2
Max. score:
6
Reversibility:
fully reversible
Irritation parameter:
overall irritation score
Remarks:
Mean Draize score
Basis:
mean
Time point:
other: 48 hrs., 72 hrs.
Score:
4.3
Max. score:
24
Reversibility:
fully reversible
Irritation parameter:
overall irritation score
Remarks:
Mean Draize score
Basis:
mean
Time point:
other: 7 day
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
4.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
cornea opacity score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
4.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
iris score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24/48/72 h
Score:
4.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
conjunctivae score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
4.5
Max. score:
6
Reversibility:
fully reversible within: 7 days
Remarks on result:
probability of weak irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.
Irritation parameter:
chemosis score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
6
Remarks on result:
no indication of irritation
Remarks:
Only total eye irritation scores were reported for individual animals from observations at 24/48/72 hours. Eye irritation was observed in 3 out of 6 animals. All eyes appeared normal by day 7 of the observation period.

The star (*) indicates the observation of a lesion

Summary of Individual Scores

 

Score

Number of Animals with Eye Response

 

Unwashed

Total

Animal Number

562

563

564

569

570

590

Ave.

Range

Observation, Prescreen

0

0

0

0

0

0

0

0

0

1 hour

4

4

14

10*

6

12*

8.3

4-12

6

24 hour

0

2

4

6*

0

0

2.0

0-6

3

48 hour

0

2

0

24*

0

0

4.3

0-24

2

72 hour

0

2

0

24*

0

0

4.3

0-24

2

4 day

0

2

0

2*

0

0

0.7

0-2

2

7 day

0

0

0

0

0

0

0

0

0

Combined average

0.7

2.0

3.0

11.0

1.0

2.0

3.3

0.7-11

 
Interpretation of results:
GHS criteria not met
Conclusions:
In this study, propane-1-thiol is an eye irritant in 3 out of 6 animals. All effects were fully reversible by day 7.
Executive summary:

In a primary eye irritation study, 0.1 millilitres of propane-1-thiol was placed on the everted lower lid of the right eye of six New Zealand white albino rabbits for seconds and then released.  The left eye remained untreated and served as a control. Eyes were not washed after exposure. Animals were observed for 7 days post dosing.  Irritation was scored by the method of Draize.

 

Ocular irritation was observed in all of the test animals following exposure (mean irritation score = 8.3, 2.0, 4.3, and 4.3 for 1, 24, 48, and 72 hours respectively). All signs of irritation dissipated at the end of the 7 day observation period.  The combined weight gain from day of dosing to day 7 was approximately 83 grams. In this study, propane-1-thiol is considered irritating to the eye based on the Draize method.

 

This study received a Klimisch score of 2 and is classified as reliable with restrictions because it complies with GLP and adheres closely to OECD guideline 405.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Draize method
GLP compliance:
no
Species:
rabbit
Strain:
other: Albino
Details on test animals or tissues and environmental conditions:
No detail available
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
Not rinsed
Observation period (in vivo):
10 min and 1, 2, 3, 4, 24, 48 and 72 hours
Number of animals or in vitro replicates:
6
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data

SCORING SYSTEM: Draize scale

TOOL USED TO ASSESS SCORE: No data
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.66
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The only effect was conjunctival inflammation with or without chemosis. Neither the cornea nor the iris was affected.
Interpretation of results:
GHS criteria not met
Conclusions:
In an in vivo eye irritation study, conducted according to a national standard method with acceptable restrictions, but prior to GLP, the test substance iso-propyl mercaptan was concluded to be not irritating to rabbit eyes.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

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

Additional information

Skin Irritation

A key primary dermal irritation study (Latven, 1976; Klimisch score = 2) was identified to evaluate the skin irritation potential of 2-methylpropane-2-thiol. In this study, six albino rabbits were dermally exposed to 0.5 mL 2-methylpropane-2-thiol under occlusive wrap for 4 hours. The animals were subsequently observed for a period of 3 days. There was no evidence of dermal irritation at any of the treatment sites and the overall irritation score (mean of 24, 48, and 72 hours) was 0. Therefore, 2-methylpropane-2-thiol was not considered to be irritating to the skin of rabbits.

Eye Irritation

In an in vitro bovine corneal opacity and permeability test study with 2-methylpropane-2-thiol, conducted according to OECD Test Guideline 437 and in compliance with GLP, an average IVIS score of 17 was obtained, which is lower that the threshold IVIS score for a category 1 substance of >55, but higher than the score of 3 indicating ‘not classified’ for eye irritation. The potential of the substance to be an ocular corrosive or severe irritant could not be established and further testing was recommended (Charles River Laboratories, 2020a).

In an in vitro eye irritation key study with the test substance 2-methylpropane-2-thiol, a reconstructed human cornea-like epithelium was used. The study was conducted according to OECD Test Guideline 492 and in compliance with GLP. The substance was applied topically on duplicate tissues and incubated at room temperature, blotted on absorbent material and incubated for another 2 hours at 5% CO2 in a humidified incubator. The cell viability was 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 % (as a reference viability).

In the preliminary tests, the substance was found to have direct MTT reducing properties but no colouring potential. The calculated mean NSMTT was 0.35% whereas the relative mean viability of the treated tissues, i.e. the true MTT metabolic conversion following correction by NSMTT was 3% ± 1% between the duplicate tissues which indicate a positive indication of eye irritation. The relative mean viability was <60% (Charles River Laboratories, 2020b). This study is not able to distinguish between Category 1 and 2, and further information is needed to conclude on classification.

The available in vitro studies conducted with the registered substance, 2-methylpropane-2-thiol indicate the potential for eye irritation but from the results it is not possible to distinguish between Category 1 and Category 2 according to the criteria set out in Regulation (EC) No. 1272/2008. Therefore, in vivo data for the structural analogues, n-propyl mercaptan (CAS 107-03-9, NPM) and isopropyl mercaptan (CAS 75-33-2; IPM) are included as part of a weight of evidence approach concluding that 2-methylpropane-2-thiol is an eye irritant (Category 2) but does not cause irreversible damage to the eyes (Category 1) in accordance with Regulation (EC) No. 1272/2008.

In an in vivo eye irritation study for the structural analogue, propane-1-thiol (NPM), conducted according to a protocol similar to OECD Test Guideline 405 and in compliance with GLP (Moon, 1981), 0.1 ml of propane-1-thiol was instilled into the conjunctival sac of the right eye of 6 young adult New Zealand white rabbits (male) for 4 seconds. The eyes were not washed subsequent to exposure and the left eyes served as control. Animals were observed for a period of 7 days post exposure and eye irritation was scored by the method of Draize. Ocular irritation was observed in all of the test animals following exposure (mean irritation score = 8.3, 2.0, 4.3, and 4.3 for 1, 24, 48, and 72 hours respectively). Ocular irritation was observed in all of the test animals at 1-hour observation. Eye irritation was observed in two out of six animals at 24/48/72 hours and one animal at 24 hours only. All signs of irritation dissipated at the end of the 7-day observation period.  At the study termination, the eyes of all animals appeared normal. Propane-1-thiol was considered to be irritating to the eyes of rabbits by the study author. However, total irritation scores of 0 at 24, 48 and 72 hours were recorded for three out of the six animals tested, therefore the results do not meet the criteria for classification of propane-1-thiol as an eye irritant.

In an in vivo eye irritation study for the structural analogue, iso-propyl mercaptan (IPM), conducted according to a national standard method with acceptable deviations, but prior to GLP (Latven, 1977), 0.1 ml of IPM was instilled into the eye of 6 white rabbits. The eyes were not washed subsequent to exposure. The animals were observed for indication of eye irritation at 10 minutes, 1, 2, 3, 4, 24, 48 and 72 hours post-instillation. Eye irritation effects were scored against the method of Draize system. The only effect was conjunctival inflammation with or without chemosis. Neither the cornea nor the iris was affected. The responses recovered within 24 hours in five of six rabbits while the sixth animal recovered within 48 hours. The test substance, IPM, was concluded to be not an eye irritant when tested in rabbit eyes.

Respiratory Tract Irritation

There are no respiratory irritation data available for 2-methylpropane-2-thiol. However, acute inhalation toxicity data available from studies conducted in rats (Daly, 1986) and mice (Fairchild and Stokinger, 1958) appear to indicate that exposure to 2-methylpropane-2-thiol does not cause respiratory irritation.

Justification for classification or non-classification

2-Methylpropane-2-thiol does not meet the criteria for classification and labelling as a skin irritant under the CLP Regulation (EC) No 1272/2008.

Based on the weight of evidence approach, 2-methylpropane-2-thiol requires classification for eye irritation Cat. 2, H319: "Causes serious eye irritation" according to Regulation (EC) No 1272/2008.

The results of two in vitro eye irritation studies with 2-methylpropane-2-thiol indicated that the substance has the potential to cause adverse effects in the eye but could not distinguish between Category 2 and Category 1 as defined in Table 3.2.3 of Regulation (EC) No 1272/2008. Further evidence from in vivo studies with two analogue substances, propane-1-thiol (NPM) and propane-2-thiol (IPM) was therefore taken in to account. A study with NPM indicated some potential for irritant effects but these were not of sufficient severity to trigger classification according to EC criteria. Likewise, IPM caused only minimal effects that did not warrant classification. It is therefore considered, based on the trend seen with analogous substances, that the registered substance is unlikely to cause irreversible damage to eyes and it is therefore classified Category 2, H319: “Causes serious eye irritation”.

2-Methylpropane-2-thiol does not require classification and labelling as a respiratory irritant under the CLP Regulation (EC) No 1272/2008.