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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
7 September 2009
Deviations:
no
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
Results were interpreted according to this updated guideline to determine GHS/CLP classification.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
other: bovine cornea
Details on test animals or tissues and environmental conditions:
The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 13.00 hours, 15 June 2010). Instructions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle. The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 15.22 hours, 15 June 2010.)

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: positive and negative substances
Amount / concentration applied:
750µl
Duration of treatment / exposure:
10 min
Observation period (in vivo):
2 h
Details on study design:
METHOD

Preparation of corneas:
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been
dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting. The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with cMEM, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders. The holders were then plugged and incubated, in an upright position, for 60 minutes ± 5 minutes at 32°C ± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout the experiment. At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM. Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Opacity measurement:
The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Once the basal opacity of all the corneas had been measured, the mean basal opacity value was calculated. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.

Test substance pH:
An estimate of the pH of the test substance was determined using pH test sticks and recorded.

Treatment groups:
Corneas were treated in triplicate with either the test substance, positive control (ethanol) or negative control (0.9% sodium chloride solution).
The controls and their results were shared with several studies performed in the same assay.

Treatment of corneas:
The test substance, Tertiododecylmercaptan, a colourless liquid, and the positive control (ethanol) were tested undiluted. Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty µl (750 µl) of test substance, positive control or negative control was introduced into the anterior part of each holder. Following application, the anterior compartment was plugged and the holder turned to a horizontal position and slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. The test material or controls were in contact with the cornea for a total of 10 minutes (± 30 seconds). Each holder was incubated in a horizontal position at 32°C ± 1°C in a waterbath.
Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed, at least three times or until the wash medium (MEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The test material, Tertiododecylmercaptan, required three washes.
The anterior compartment was then filled with cMEM taking care to ensure no air bubbles were present in the compartment. Once all the air bubbles had been removed, the anterior compartment was re-plugged and the holders returned to the waterbath and incubated, in an upright position, for two hours ± 10 minutes at 32°C ± 1°C.
Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.
Throughout the assay the corneas were examined for opaque spots or other irregularities.

Permeability determination:
The sodium fluorescein stock solution (4 mg/ml) was thawed prior to commencing the permeability incubation. Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One ml (1ml) of the sodium fluorescein solution was added to the anterior compartment using a micropipette. Following addition of the sodium fluorescein solution to the anterior side of the holder, the compartment was plugged and the corneas incubated in a horizontal position at 32°C ± 1°C for 90 ± 5 minutes in a waterbath.
Following incubation, the medium in the posterior compartment was mixed by drawing approximately 2.5 ml gently up and down a 5 ml syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 490nm (OD490) and a sample of cMEM read (OD = 0.061). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution giving an OD490 value above 1.8 was diluted 1 in 5 with cMEM.

Criteria for determination of a valid test:
The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean for the laboratory. The negative control mean opacity change value should be 2.0 and the permeability mean value 0.1.

ASSESSMENT OF RESULTS

Opacity:
The change in the opacity of each cornea was calculated by subtracting the initial basal opacity from the post-treatment opacity measurement. The mean change in opacity for the negative control corneas was calculated and was subtracted from the change in opacity of each treated cornea to obtain the corrected opacity value. The mean corrected opacity change value of each treatment group (of three corneas) was calculated from the individual corrected opacity values of the treated corneas.

Permeability (OD490):
The corrected permeability value (OD490) of each treated cornea was calculated by subtracting the mean negative control cornea value from the permeability value of each cornea. The mean corrected permeability value of each treatment group was calculated from the individual corrected permeability values of the treated corneas.

In Vitro Irritancy score calculation:
The In Vitro Irritancy Score (IVIS) was calculated using the following formula:
In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490 Value)
The IVIS was calculated for each individual treatment and positive control cornea. The mean IVIS value for each treatment group was calculated from the IVIS of each individual cornea in the treatment group.

Classification criteria
A substance that induces an IVIS 55.1 is defined as a corrosive or severe irritant. If the test substance is not identified as an ocular corrosive or severe irritant, additional testing (not part of this study) should be conducted for classification or labelling purposes.
IVIS In Vitro Classification
55.1 Corrosive/Severe irritant
< 55.1 Non-Corrosive/Severe irritant

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
-0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
-0.333
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Value:
-0.013
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Assay validity:
The positive control, ethanol, elicited an In Vitro Irritancy Score of 45.4. This value was within the historical range (mean ± 2 x SD = 31.3 – 63.1) for the assays performed to date. The negative control, 0.9% saline, opacity mean change value was 0.000 which was below the maximum acceptance value of 2.0. The permeability mean of the negative control was 0.021 which was below the maximum acceptance value of 0.1.

pH determination:
The pH of the test substance, measured using pH sticks, was approximately 7.0.

BCOP results:
Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with the test substance, Tertiododecylmercaptan, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas treated with the negative control, 0.9% saline, were clear. The results of the BCOP assay are summarised in the enclosed table.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Tertiododecylmercaptan, elicited an In Vitro Irritancy Score of -0.5 ± 0.6 and was predicted to be a non eye irritant.
Executive summary:

The potential ocular irritancy of t-dodecyl mercaptan was assessed in vitro in the Bovine Corneal Opacity and Permeability Assay (BCOP) performed following OECD guideline #437 and GLP. Ethanol was tested in parallel as a positive control. The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which can be used to classify and rank test substances as potential eye irritants.Tertiododecylmercaptan, elicited an In Vitro Irritancy Score of -0.5 ± 0.6 and was predicted to be a non eye irritant.