2,2'-[1,2-ethanediylbis(oxy)]bis(ethanethiol)

Endpoint summary

Description of key information

Skin irritation

OECD 439

The potential of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE (DMDO) to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™ (de Marzy, 2016). The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. DMDO strongly interacts with MTT. These results do not allow a proper assessment of the irritation properties of the test item, indicating that the test method is not suitable for its hazard identification.

OECD 402

The acute toxicity of DMDO was investigated following dermal administration of a single dose to the rat (Caroso, 2016). A single dose of 2000 mg/kg was administered to a group of 5 male and 5 female animals for 24 hours. After 14 days, all animals were killed and subjected to necropsy examination. No skin irritation was observed at the treated site.

Eye irritation

OECD 437

The corneal damage potential of DMDO was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea (Andres, 2017). DMDO was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 +/- 1°C for 1 hour and whose opacity had been determined. DMDO was incubated on the cornea for 10 minutes at 32 +/- 1°C. After removal of the test item and 1 hour and 55 minutes post-incubation, opacity and permeability values were measured. The negative control (HBSS-solution) and the positive control (undiluted dimethylformamide) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid. DMDO was tested as pure. Under the condition of this test , DMDO showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 4.75. According to the OECD Guideline no. 437 (July 2013), a substance with an IVIS >3 and <= 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage. Nevertheless, the result did not warrant a classifiucation as eye damage category 1.

OECD 405

The acute eye irritation of DMDO was investigated in rabbits following the OECD test guideline no. 405 (Di Manno, 2017). A 0.1mL aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours after dosing.

No irritation at either the conjunctivae, iris or cornea was recorded in any treated animal during the whole observation period of the study. There were no significant signs of pain/distress after dosing. Changes in body weight were not remarkable. There was no indication of a systemic effect related to treatment. These results indicate that the test item, DIMERCAPTO-1,8-DIOXA-3,6-OCTANE, has no effect on the eye of the rabbit.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)

Reason / purpose for cross-reference:

reference to other study

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 24 June 2016; End of experimental phase: 08 August 2016. Study completion:15 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

DMDO is a strong MTT reducer, this method is not suitable for its hazard identification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted on 28 July 2015

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from a single donor

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

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).

Duration of treatment / exposure:

An exposure time of 15 ± 0.5 minutes was allowed in a ventilated cabinet at room temperature.
Due to the non uniform distribution of the test item on the epidermidis, a redistribution on the treated tissues after an exposure time of 7 minutes was performed.

Duration of post-treatment incubation (if applicable):

A 42 ± 1 hour recovery period was allowed by incubation at 37°C, 5 % CO2 and saturated humidity.

Number of replicates:

Negative control (live tissue): 3 replicates
Positive control (live tissue): 3 replicates
Test item (live tissue): 3 replicates
Negative control (killed tissue): 2 replicates
Test item (killed tissue): 2 replicates

Remarks on result:

other: DMDO is a strong MTT reducer, this method is not suitable for its hazard identification.

Other effects / acceptance of results:

The mean cell viability of the test item, with only OD-blank subtraction, was 69 % of the concurrent negative control value, with an acceptable variability between replicates (SD of % viability = 6.3 lower than 18); however, the NSMTT control was 110 %, higher than the acceptability criteria stated in the OECD guideline n.439.

Interpretation of results:

study cannot be used for classification

Conclusions:

The potential of the test item to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The blank, negative and positive controls gave acceptable results and the study was accepted as valid. Since the test item is a strong MTT reducer, this method is not suitable for its hazard identification.

Executive summary:

The potential of DMDO to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A dark violet solution was noticed at the end of the incubation period, indicating that the test item can interact with MTT. Based on this result, an additional control was added in the Main Assay. In a second step, the test item was assayed for the ability of colouring water per se. No colour change was observed; spectral analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 0.068, indicating that the test item does not have a potential interfering ability. 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), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 µL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (10.0 % of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.5). Based on the stated criteria (mean viability = 40% and SD of % viability = 18), the assay was regarded as valid. The mean cell viability of the test item was 69 %, with an acceptable variability between replicates (SD of % Variability = 6.3). However, the NSMTT was 110 % of the concurrent negative control with alive tissues. This result indicated that the test item strongly interacts with MTT. These results do not allow a proper assessment of the irritation properties of the test item, indicating that the test method is not suitable for its hazard identification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2016 (Study Plan dated) to 30 January 2017 (Final Report dated)

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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Species:

other: Bos primigenius taurus

Details on test animals or tissues and environmental conditions:

Isolated corneas from the eyes of freshly slaughtered bovine (age of animal: 12-60 months)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 microliter

Duration of treatment / exposure:

10 minutes at 32°C +/- 1°C

Duration of post- treatment incubation (in vitro):

1 hour and 55 minutes at 32°C +/- 1°C

Number of animals or in vitro replicates:

3 fresh bovine corneas

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing
- Time after start of exposure:10 minutes

SCORING SYSTEM: Final opacity value of each cornea
Fluorescein permeability after 85 minutes of incubation at 32 +/- 1°C

TOOL USED TO ASSESS SCORE: opacitometer (opacity), spectrophotometer at 492 nm (permeability)

Irritation parameter:

in vitro irritation score

Value:

ca. 4.75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS = 0.12

Positive controls validity:

valid

Remarks:

IVIS = 82.08

Interpretation of results:

other: Not classified eye damage category 1

Conclusions:

The test item DMDO induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage

Executive summary:

The study was performed to assess corneal damage potential of DMDO by quantitative measurements of changes in opacity and permeability in a bovine cornea. DMDO was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 +/- 1°C for 1 hour and whose opacity had been determined. DMDO was incubated on the cornea for 10 minutes at 32 +/- 1°C. After removal of the test item and 1 hour and 55 minutes post-incubation, opacity and permeability values were measured. The negative control (HBSS-solution) and the positive control (undiluted dimethylformamide) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid. DMDO was tested as pure. Under the condition of this test , DMDO showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 4.75. According to the OECD Guideline no. 437 (July 2013), a substance with an IVIS >3 and <= 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage. Nevertheless, the result did not warrant a classifiucation as eye damage category 1.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 13 March 2017 End of experimental phase: 17 march 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

adopted on 2 October 2012.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Species and strain: Rabbit, New Zealand White Specific Pathogen Free (SPF)
Sex: Females
Age at the start of study: 13 weeks old
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’Arbresle Cedex, France
Date of arrival: 19 January 2017
Acclimatisation period: At least 14 days
Veterinary health check: After arrival

ENVIRONMENTAL CONDITIONS
Animals per cage: Up to 2 during acclimatisation; 1 during the study
Housing: Polycarbonate/stainless steel cages measuring 65.3×65.3×45 cm with perforated NorylTM floor suspended over trays
Cage control: Daily inspected and changed as necessary (at least 3 times/week)
Water Drinking: water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 2RB15,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese
(MI)
Diet supply ad libitum throughout the study
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 19 °C±2 °C
Relative humidity: range 55%±15%

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1mL aliquot of the test item onto the right eye.

Duration of treatment / exposure:

Animals were dosed once only

Observation period (in vivo):

The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after the end of dosing. At the same time intervals, animals and cages were inspected for evidence of clinical signs indicating a potential systemic effect. Signs of pain/stress of the animals were evaluated two times on the day of dosing, an additional observation was performed at the end of working day, then once daily at approximately 24, 48 and 72 hours after treatment.

Number of animals or in vitro replicates:

3 female animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No rinsing of the treated eye was performed after dosing.
- Time after start of exposure:The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after the end of dosing.

SCORING SYSTEM:
Conjunctivae

A) Reddening of the palpebral and bulbar conjunctivae
0 = Blood vessels normal
1 = Some blood vessels definitely hyperaemic (injected)
2 = Diffuse, crimson colour, individual vessels not easily discernible
3 = Diffuse, beefy red

B) Chemosis of the lids and/or nictitating membrane
0 = No swelling
1 = Any swelling above normal (including nictitating membrane)
2 = Obvious swelling with partial eversion of the lids
3 = Swelling with lids about half closed
4 = Swelling with lids more than half closed

C) Discharge (Lachrymation)
0 = No discharge
1 = Any amount different from normal (does not include small amount observed in inner canthus of normal animals)
2 = Discharge with moistening of the lids and hair just adjacent to the lids
3 = Discharge with moistening of the lids and hair for a considerable area
around the eye

Iris
0 = Normal
1 = Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 = No reaction to light, haemorrhage, gross destruction (any or all of these)

Cornea
A) Degree of opacity (area most dense taken for reading)
0 = No ulceration or opacity
1 = Scattered or diffuse area of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2 = Easily discernible translucent area, details of iris slightly obscured
3 = Nacreous area, no details of iris visible, size of pupil barely discernible
4 = Opaque cornea, iris not discernible through the opacity

B) Area of cornea involved
1 = One quarter (or less) but not zero
2 = Greater than one quarter, but less than half
3 = Greater than half, but less than three quarters
4 = Greater than three quarters, up to whole area

TOOL USED TO ASSESS SCORE: Macroscopic observation with the aid of light pen

Irritation parameter:

cornea opacity score

Basis:

animal: #1, 2 and 3

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, 2 and 3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: #1, 2 and 3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: #1, 2 and 3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

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

Other effects:

There was no indication of a systemic effect.
No signs of pain or distress were observed during the study.
Changes in body weight, seen during the course of the study, were not remarkable.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute eye irritation of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE was investigated in the rabbit.
No irritation was recorded in any treated animal during the observation period.

Executive summary:

The acute eye irritation of DIMERCAPTO-1,8-DIOXA-3,6-OCTANE was investigated in rabbits following the OECD test guideline no. 405. A 0.1mL aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours after dosing.

No irritation at either the conjunctivae, iris or cornea was recorded in any treated animal during the whole observation period of the study. There were no significant signs of pain/distress after dosing. Changes in body weight were not remarkable. There was no indication of a systemic effect related to treatment. These results indicate that the test item, DIMERCAPTO-1,8-DIOXA-3,6-OCTANE, has no effect on the eye of the rabbit.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Not classified according CLP and GHS criteria.