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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 June to 19 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study caried out in accordance with internationally recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
460-230-6
EC Name:
-
Cas Number:
36484-54-5
IUPAC Name:
Oligomeric reaction products of 4,4'-propane-2,2-diyldiphenol and 2-methyloxirane and 2-(chloromethyl)oxirane
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Aliphaticdiol diglycidyl ether
- Physical state: liquid
- Analytical purity: 99%
- Lot/batch No.: 20481
- Expiration date of the lot/batch: 01 April 2018

Test animals / tissue source

Species:
other: isolated bovine corneas
Strain:
other:
Details on test animals or tissues and environmental conditions:
The bovine eyes, supplied by an abattoir, were excised and collected as soon after slaughter as possible. Eyes were obtained from cattle aged less than 30 months. 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.

Test system

Vehicle:
other: 0.9% saline.
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 750 µL- Concentration (if solution): 10% v/v in vehicle
Duration of treatment / exposure:
10 minutes (± 30 seconds)
Observation period (in vivo):
2 hours ±10 minutes (opacity) then 90 ± 5 minutes for permeability
Number of animals or in vitro replicates:
Corneas treated in triplicate
Details on study design:
REMOVAL OF TEST SUBSTANCE - Washing:
Following incubation, the test substance, positive and negative controls were removed from the epithelial surface of the cornea by washing, at least three times or until the wash medium (Eagles Minimal Essential Medium with phenol red) was clear and there was no discolouration. The corneas were gently rinsed with the wash medium until the medium was clear and the colour unchanged. The test substance proved difficult to remove by this method, so the front glass was removed from the holder and the cornea carefully washed using a gentle stream of Minimal Essential Medium with phenol red from a syringe.

SCORING SYSTEM:
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. 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.

Permeability: Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One mL of sodium fluorescein solution was added to the anterior compartment using a micropipette.and the compartment 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 490 nm (OD490) and a sample of cMEM read (OD = 0.079). 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 complete Minimal Essential Medium.

The In Vitro Irritancy Score (IVIS) was calculated by combining the coeneal opacity and permeability using the following formula:

IVIS = Corrected Opacity Value + (15 x Corrected Permeability 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.


TOOL USED TO ASSESS SCORE: spectrophotometer adjusted to read at 490 nm

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
EP-4000S
Value:
-0.4
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Sodium Hydroxide 1% (w/v)
Value:
131.4
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

The pH of the test substance, Aliphaticdiol diglycidyl ether, as 10% (v/v) suspension in 0.9% saline, was approximately 7.5.

Throughout the assay the corneas were examined for opaque spots or other irregularities. Following treatment with test substance, Aliphaticdiol diglycidyl ether, 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 table below.

Sample

Opacity
 ± SD

Permeability
± SD

In vitro irritancy

Score
 ± SD

Aliphaticdiol diglycidyl ether

1.000 ± 2.082

0.007 ± 0.015

1.1 ± 2.3

Ethanol

28.333 ± 1.732

1.146 ± 0.183

45.5 ± 2.7

0.9% Saline

0.667 ± 0.577

0.005 ± 0.002

Not applicable

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU