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Diss Factsheets

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
Study period:
2010-07-27 TO 2010-08-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed following GLP and performed following the Draft Standard Operating Procedure of MatTek Corporation -Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm).

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other:
Deviations:
not applicable
Principles of method if other than guideline:
Study was performed following GLP and performed following the Draft Standard Operating Procedure of MatTek Corporation -Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm).
GLP compliance:
yes (incl. QA statement)
Remarks:
Hameln rds a.s., Section of Biological studies, Horna 36, 900 01 Modra, Slovak Republic.

Test material

Constituent 1
Reference substance name:
Distillation residue, butyl alcohols production, rectification
EC Number:
931-740-0
Molecular formula:
undefined (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Distillation residue, butyl alcohols production, rectification
Details on test material:
Composite sample K-303·111. A composite sample, consisting of 5 sub-samples taken over a period of 3 consecutive days, was collected directly from the process line. The volume of each sub-sample was approximately 5-10 L. The sub-samples were combined, resulting in a total volume of ca. 50 L. This ca. 50 L composite sample was split into two equal portions of ca. 25 L each (control sample and laboratory sample). The ca. 25 L laboratory sample was further split into two portions of ca. 5 L and 20 L before sending for analysis at two separate laboratories. After sampling and sample splitting, all the portions of the composite sample were stored at +4 deg Celsius.

Test animals / tissue source

Species:
other: human corneal model
Strain:
not specified

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
50 microliters topically applied
Details on study design:
Test system
specific model for eye irritation testing EpiOcularTM (OCL-200) (Lol No. 13059) was obtained from MatTek Corporation. USA. OeL-200 kit was supplied by the MatTek Corporation instead of ordered OCL-21 2 kit (12 tissues). The difference between these two kits is only in the number of tissues.

Control material
Concurrent negative and positive controls were used in experiment to ensure adequate performance or lhe experimental model.
Negative control (NC); Sterile deionised water
Positive control (PC); Ethyl acetate, p.a.

Chemicals, media
Assay Medium OCL- IOO·ASY (Dulbecco's Modified Eagles medium - DMEM) Lot 072310A IIB (MatTek). Phosphate Buffered Saline (PBS) Lot 28104117 (Flow Laboratories). Elhyl acetate p.a. Lot. 1523872008 (Merck). MTT (3-[4 ,5-Dimethylhiazol·2·yI]-2 ,5-diphenyltetrazolium bromide) 98% Lot. 51K5312 (Sigma). isopropanol p.a. Lot. A090313/01 (Mikrochem), sterile deionized Water, tissue culture grade (hameln rds a.s.).

Protocol
In vitro eye irritation: specific human corneal model test was used. Test was performed in compliance with Draft Standard Operating Procedure of MatTek Corporation - Eye Irritation Validation Study (EIVS): Ocular Irritation Assay for Chemicals using EpiOcular (tm) and in compliance with Principle GLP Section of Biological Studies (2008). Two tissue replicates were used for each treatment (exposure time), including controls. Protocol for liquid test articles was used: test product was tested by applying of 50 microliters topically on cultures for 30 minutes, followed by a 12-minute post-trealment immersion and a 120-minute post-treatment incubation. The tissues were incubated at standard culture conditions (37 ± 1°C) in a humidified atmosphere of 5 ± 1 % C02 in air). After exposure period (30-min). each tissue will be rinsed gently with PBS to remove any residual test article. The magnitude of viability was quantified by using MTT test (SOP I).

Test article treatment
After the 30-minute pre-treatment with PBS. test product (concentrated) was dispensed directly atop the EpiOcular™ tissue at volume of 50 microliters. After exposure period (30-min), each tissue was rinsed extensively with PBS to remove any residual test article (three clean beakers containing 100 mL each of PBS were used).

Test for direct MTT reduction
A test substance may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present in the tissues when the MTT viability test is performed. To identify this possible interference of test product. this test was performed in the previous study (4).

Receipt and preparation of cultures
EpiOcularTM was delivered one day prior or experiment performing. Each culture was removed with sterile forceps from the agarose gel, inspected, and transferred to a pre-labeled 6-well plates containing 1 mL of assay medium per well. The EpiOcular™ cultures were incubated at 37 ± 1°C in a humidified atmosphere of 5 ± I % C02 in air for overnight prior to dosing for release of transport stress related compounds and debris.

Assay procedure
After overnight pre-incubation tissues were pre-treated with 20 mL of PBS without calcium and magnesium. The tissues were incubated at standard culture condition for 30 minutes. Then, 50 microliters of the test or control articles were applied topically onto the tissue surface for 30 minutes. Two tissues each were used per treatment, negative and positive control. The cultures were returned to the incubator for the appropriate exposure times. After this, the test and control articles were extensively rinsed from the cultures using PBS without calcium and magnesium. After rinsing, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed Assay medium in a prelabelled 12-well plate for a 12 min immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, each insert was transferred to the appropriate well of the prelabelled 6-well plate containing 1 mL of Assay medium. The tissues were incubated for 120 min at standard culture conditions (Post-treatment incubation). After this, the cultures were transferred to 24-wells plate containing 0.3 mL/well of MTT reagent (1 mg/mL) and incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air for 3 hours.After incubation, the cultures were blotted on absorbent paper and extracted in 2 mL of isopropanol overnight without shaking at room temperature. At the end of the extraction period, the liquid with in each insert was decanted into the well from which it was taken. The extract solution was mixed for 15 min and two 200 microliters aliquots were transferred to a 96-well plate and the absorbances were recorded.

Treatment of results
Data are included cytotoxicity and viability determination. The optical densities (ODs) were read in a 96-well plate spectrophotometer using a wavelength 540nm without a reference filter. Data of optical densities generated by microplate reader and already corrected by substraction of the blanks mean from all OD values were manually transported into the first map (Import) of the EXCEL spreadsheet in the 96-well format. The second sheet (Results) were made calculations and provided a column graph of the results. The OD values obtained for each test sample were used to calculate a percentage viability relative to negative control. which is arbitrarily set at 100 %. All data were summarised in tabular form in protocol (1, 2).

Assay acceptance criteria
Negative control:
The absolute optical density (OD) of the H2O treated NC tissues in the MTT-test is an indicator of tissue viability obtained under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the mean of NC is OD>= 1.
Positive control:
30-min exposure of the PC must reveal a mean tissue viability less than 60 %.

Evaluation criteria
The prediction of eye irritation associated with EpiOcular™ model is:
• If the test article-treated tissue viability is > 60% relative to negative control-treated tissue viability, the test article is labeled non-irritant.
• If the test article-treated tissue viability is < 60% relative to negative control-treated tissue viability, the test article is labeled irritant

Results and discussion

In vivo

Results
Irritation parameter:
other: MTT reduction score
Basis:
mean
Time point:
other: 120 minutes
Score:
>= 27 - < 28.8
Max. score:
100
Reversibility:
not specified
Remarks on result:
other: Threshold is 60%, if below than considered as irritant to eye

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance was examined for in vitro eye irritation in human corneal model test EpiOcular(tm). Validity of the test method was ascertained by positive control ethyl acetate. Two tissue replicates were used for each treatment (exposure time 30-min, including controls). The magnitude of viability was quantified by using MTT-test. Determined viability of culture treated with substance fulfilled criteria for eye irritation test. It has been concluded that substance is
considered to be irritant to eye.