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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
Study period:
2015-10-22 - 2015-12-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented OECD GLP guideline study without deviations on the registered substance itself.

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)
Version / remarks:
OECD Guideline for Testing of Chemicals 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 (July, 2013).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-methylenebis[6-cyclohexyl-p-cresol]
EC Number:
223-773-8
EC Name:
2,2'-methylenebis[6-cyclohexyl-p-cresol]
Cas Number:
4066-02-8
Molecular formula:
C27H36O2
IUPAC Name:
2-cyclohexyl-6-[(3-cyclohexyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol
Test material form:
solid: particulate/powder
Details on test material:
- Substance type: pure substance
- Storage condition of test material: At room temperature, protected from light

Test animals / tissue source

Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml (corresponds to approx. 150 mg)
- Concentration (if solution): 20% suspension

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 ml suspension (corresponds to approx. 0.6 ml)
- Concentration (if solution): 0.9% (w/v) NaCl (saline)
Duration of treatment / exposure:
240 min
Observation period (in vivo):
no post-observation
Number of animals or in vitro replicates:
each 3 corneae for test group, positive and negative control
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with saline
- Time after start of exposure: 240 min

SCORING SYSTEM: IVIS = opacity value + (15 x OD490 value), In vitro Irritancy Score (according to OECD 437)
≤ 3 : No Category (according to GHS)
> 3; ≤ 55 : No prediction can be made
> 55 : Serious eye damage according to CLP/EPA/GHS (Cat 1)

TOOL USED TO ASSESS SCORE: opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) / fluorescein + spectrophotometer (Versamax® Molecular Devices)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
test item
Run / experiment:
mean, 240 min
Value:
5.31
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
yes, vehicle
Positive controls validity:
valid
Remarks on result:
other: test item
Irritation parameter:
in vitro irritation score
Remarks:
negative control
Run / experiment:
mean, 240 min
Value:
1.14
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
negative control, not categorized
Irritation parameter:
in vitro irritation score
Remarks:
positive control
Run / experiment:
mean, 240 min
Value:
127.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
positive indication of irritation
Remarks:
positive control, Category 1

In vivo

Irritant / corrosive response data:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.14).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity of the corneae (mean IVIS = 127.14) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item caused a slight increase of the corneal opacity. Permeability did not occur. The calculated mean IVIS was 5.31 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 no prediction for the damage hazard of the test item to the eye can be made.

Applicant's summary and conclusion

Interpretation of results:
other: not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS)
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
Testing was performed via a GLP OECD 437 guideline study on the registered substance itself. The present in vitro method is recommended in a tiered testing approach and the validity criteria are met, making the results sufficiently reliable to assess the corrosivity of 2,2'-Methylen-bis-(4-methyl-6-cyclohexylphenol) to the eye. The present in vitro method allows the identification of corrosive chemical substances and mixtures or of substances which do not require classification.
According to OECD Guideline no. 437 (Jul. 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.
Hence, no conclusion can be drawn whether the substance needs to be classified as eye irritant, for a clear distinction an in vivo study would be required based on the current state of the art.
According to the OECD 437guideline, “Test chemicals inducing serious eye damage are classified as UN GHS Category 1. Chemicals not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category“.
Based on the tonnage-driven data requirements under REACH (Annex VIII), the present result “inconclusive” may not serve as a stand-alone result. As, according to the present results, no prediction can be made with regard to the classification of 2,2'-Methylen-bis-(4-methyl-6-cyclohexylphenol), but it is evident that the substance does not need to be classified as UN GHS Category 1, it will be classified in a precautionary approach as UN GHS Category 2, as no additional testing is intended due to animal welfare.
Executive summary:

This in vitro study according to OECD guideline 437 under GLP was performed to assess the corneal damage potential of 2,2'-Methylen-bis-(4-methyl-6-cyclohexylphenol) by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed distinct opacity of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, 2,2'-Methylen-bis-(4-methyl-6-cyclohexylphenol) caused a slight increase of the corneal opacity, permeability effects were not observed compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 5.31. According to OECD 437, the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted. So, due to animal welfare, the substance will be classified as UN GHS Category 2.