(R*,R*)-1,4-dimercaptobutane-2,3-diol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

3 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

Amendment Commission Regulation (EC) No 2017/735, dated 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

EC Number:

222-468-7

EC Name:

(R*,R*)-1,4-dimercaptobutane-2,3-diol

Cas Number:

3483-12-3

Molecular formula:

C4H10O2S2

IUPAC Name:

1,4-disulfanylbutane-2,3-diol

Specific details on test material used for the study:

Batch-No.: 44606300 / 50774
Storage: refrigerator, 2-8°C, under Nitrogen

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 ºC to 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03g

POSITIVE AND NEGATIVE CONTROL
-Positive control: 0.03g
-Negative control: 30uL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Test item and positive control samples: Three replicates
Negative control samples: One replicate

Details on study design:

DETAILS ON TEST SYSTEM
- Strain of chicken: ROSS 308
- Supplier: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Handling and Storage: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 ºC to 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, it is used as part of a tiered testing strategy for regulatory purposes. In this assay, test items that are positive can be classified as ocular corrosives or severe irritants (UN GHS Category 1) while clearly negative test items can be identified as not requiring classification and labelling (UN GHS No Category) without further testing.

DETAILS ON THE TEST PROCEDURE USED
- Eyes selection: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

- Preparation: The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

- Acclimatization: The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

-Baseline assessment:
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0% to 2%) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

-Application: After the zero reference measurements in each experiment, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the center of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test item, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.
The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 µL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

-Exposure: 10 seconds

-Rinsing: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.

- Post-rinsing observation period: The gentle rinsing with 20 mL saline was performed in all test item and positive control treated eyes after the 30, 75, 120 and 180 minutes of observation.

- Observation and assessment of corneal effects: The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable. The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

- Histopathology: A histopathology of the corneas was not performed as not borderline results were obtained, for which histopathology could provide final clarification. Corneas are discarded 2 months after the final report.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 75 min

Run / experiment:

1

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean maximum corneal swelling up to 75 min: 0 %

Positive controls validity:

valid

Remarks:

mean maximum corneal swelling up to 75 min: 33 %

Remarks on result:

other: ICE Class II

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

percent corneal swelling upt to 240 min

Run / experiment:

1

Value:

9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean maximum corneal swelling upt to 240 min: 0 %

Positive controls validity:

valid

Remarks:

mean maximum corneal swelling upt to 240 min: 40 %

Remarks on result:

other: ICE Class II

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum corneal opacity

Run / experiment:

1

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean maximum corneal opacity: 0.0

Positive controls validity:

valid

Remarks:

Mean maximum corneal opacity: 4.0

Remarks on result:

other: ICE Class III

Results 4

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

1

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean fluorescein retention: 0.0

Positive controls validity:

valid

Remarks:

Mean fluorescein retention: 3.0

Remarks on result:

other: ICE Class III

Other effects / acceptance of results:

ACCEPTANCE OF TEST RESULTS
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
Positive and negative control values were within the corresponding historical control data ranges.

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Remarks:

The study is used for classification in a weight of evidence approach.

Conclusions:

In this ICET, the overall ICE classes were once II and twice III. According to the guideline OECD 438, 1,4-Dithiothreitol (CAS 3483-12-3) is categorized as “No prediction can be made”.

Executive summary:

In this ICET, the overall ICE classes were once II (based on the corneal swelling of 9% within 240 minutes) and twice III (based on the corneal opacity score of 2.0 and fluorescein retention of 2.0).

The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative controls were in the historical control range. So, the positive and negative controls showed the expected results. The experiment was considered to be valid.

In this ICET, the overall ICE classes were once II and twice III. According to the guideline OECD 438, 1,4-Dithiothreitol (CAS 3483-12-3) is categorized as “No prediction can be made”.