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Administrative data

Description of key information

Skin irritation: irritating | Human Skin Model | OECD 439 |  
Eye irritation: irritation score >3 - <55 | Bovine Corneas | OECD 437 |

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ISO 10993-10 Annex D: 2010 "Biological evaluation of medical devices - Part 10"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
EPISKIN-SM (SkinEthic) (TM)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK)
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
10 µL (26.3 µL/cm2) direct application of undiluted test item atop the EPISKIN-SM (TM) tissue.
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
test item 15 min treatment
Value:
11
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0
Positive controls validity:
valid
Remarks:
14.0
Other effects / acceptance of results:
The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
The mixture of 10 µL of the test item per 90 µL aqua dest. showed no colouring detectable by unaided eye-assessment.

TEST RESULTS

The test item showed irritant effects. The mean relative tissue viability (% negative control) was <= 50 % (11 %) after 15 min treatment and 42 h post incubation.

The controls confirmed the validity of the study. The mean OD550 of the six blank values was< 0.1. The mean absolute OD550 of the three negative control tissues was >= 0.6 and <= 1.5. The mean relative tissue viability (% negative control) of the positive control was <= 40 % (14%). The maximum standard deviation of viability of replicate tissues of all dose groups was < 18 % (2.4 % - 11.0 %).

Negative Control

Positive Control

Test Item

mean OD550 (3 tissues)

1.057 *

0.148

0.117

SD OD550

0.104

0.029

0.051

mean relative tissue viability [%]

100.0

14.0 **

11.0

SD tissue viability [%] ***

11.0

2.4

5.4

CV [% viability]

11.0

17.0

48.6

* Corrected mean OD550 of the negative control corresponds to 100 % absolute tissue viability.

** Mean relative tissue viability of the 3 positive control tissues is <= 40 %.

*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18 %.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed irritant effects. The test item is therefore classified as "irritant" in accordance with UN GHS "Category 2".
Executive summary:

SUMMARY

In the present study the skin irritant potential of 3,4-Epoxycyclohexylmethyl methacrylate was analysed. The EPISKIN-Standard Model (EPISKIN-SM (TM)), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS "Category 2" skin irritating test substances and not categorized test substances ("No Category") which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 15 min of exposure and 42 h post incubation was <= 50 %. The test item is therefore classified as "irritant" in accordance with UN GHS "Category 2".

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine Corneas
Strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL
Duration of treatment / exposure:
10 min
Duration of post- treatment incubation (in vitro):
- 2 h (opacity measurement)
- 3.5 h total (determination of optical density)
Number of animals or in vitro replicates:
3 corneas
Details on study design:
- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive control treated with ethanol 100%
- The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean test item
Value:
10.16
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS 2.08
Positive controls validity:
valid
Remarks:
IVIS 86.02

RESULTS

 Cornea No.  Test Item  Corr. Opacity  Corr. OD490  IVIS
 1  Neg. Control  0.00  0.027  
 2  Neg. Control  4.00  0.021  
 3  Neg. Control  1.00  0.035  
 mean  Neg. Control  1.67  0.028  2.08
 4  Pos. Control  50.33  1.919  
 5  Pos. Control  64.33  2.053  
 6  Pos. Control  53.33  2.031  
 mean  Pos. Control  56.00  2.001  86.02
 7  Test Item  13.33  -0.001  
 8  Test Item  9.33  -0.022  
 9  Test Item  8.33  -0.012  
 mean  Test Item  10.33  -0.011  10.16

The following mean in vitro irritation score was calculated: 10.16

No prediction can be made regarding the classification of the test substance 3,4-Epoxycyclohexylmethyl methacrylate according to the evaluation criteria. Further testing in another suitable method is required.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

HISTOPATHOLOGY

Negative control samples did not show any histological lesions.

In positive control samples, the main lesions consisted of vacuolated corneal epithelia and single cell apoptosis. In one sample (PK1), there was a marked epithelial flattening/height reduction associated with a focal chronic injury. This corneal sample is deemed to be affected before study start by an inflammatory/degenerative that is supported by the presence of brownish pigment (hemosiderin). In sample PK 3, there was a slight laceration of the cuboidal basal cell layer.

In test item treated samples, there was slight multifocal apoptosis/necrosis in the epithelium of all specimens.

Interpretation of results:
other: Irritation score of 10.16 and histopathological findings indicate slight signs of irritation
Remarks:
Criteria used for interpretation of results: BCOP standard evaluation criteria
Conclusions:
No prediction can be made regarding the classification of the test substance 3,4-Epoxycyclohexylmethyl methacrylate according to the evaluation criteria. Further testing in another suitable method is required.
Under the conditions of this study, 3,4-Epoxycyclohexylmethyl methacrylate caused indicators for irritation consisting of slight multifocal apoptosis/necrosis associated with multifocal vacuolation in the epithelium of all specimen.
Executive summary:

The eye irritancy potential of 3,4-Epoxycyclohexylmethyl methacrylate was investigated in the bovine corneal opacity and permeability assay.

- Preparation of the test item: tested as provided by the sponsor

- Mean in vitro irritation score: 10.16

- Classification: No prediction can be made

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In a two- step procedure (top- down approach), 3,4-epoxycyclohexylmethyl methacrylate was tested in vitro for skin corrosion and skin irritation. The first test, an OECD TG 431 study (GLP, Klimisch 1), revealed no corrosive potential of the test item. According to the second test, an OECD TG 439 (GLP, Klimisch 1), 3,4-epoxycyclohexylmethyl methacrylate has to be classified as irritant (GHS Skin Irrit. 2). Further information on the skin irritation potential can be obtained from the LLNA study conducted with mice. In this test, topical application of undiluted 3,4- epoxycyclohexylmethyl methacrylate led only to slight signs of irritation.

Eye Irritation

As first step, data available for structural homologues to 3,4-epoxycyclohexylmethyl methacrylate were evaluated. Both 1,2-epoxycyclohexane and methacrylic acid represent structural elements which are also contained in 3,4-epoxycyclohexylmethyl methacrylate. They are considered to be corrosive substances which in consequence bear also an eye damaging potential. The same conclusion was drawn for 2,3-epoxypropyl methacrylate, which contains glycidol instead of epoxycyclohexanol as alcoholic component. However, a corrosive potential did not become evident in the case of 3,4-epoxycyclohexylmethyl methacrylate on the basis of in vitro tests on skin irritation/corrosion and measurements of the skin irritation potential in the LLNA. Therefore, a conclusion to structural homologues with regard to irritating/corrosive effects seemed not to be appropriate. Nevertheless, the evidence from structural homologues supported the decision for a top-down approach to evaluate the eye irritation potential of 3,4-epoxycyclohexylmethyl methacrylate.

The BCOP was chosen as test system, since an OECD guideline is available for this method, and furthermore it addresses membrane lysis, which is the proposed mode of action for esters such as 3,4-epoxycyclohexylmethyl methacrylate (Scott, 2010). As an epoxide function is contained in the molecule as well, which is expected to induce actions on macromolecules, additional histological examinations were included in the BCOP test (Scott, 2010). The BCOP test was conducted according to OECD TG 437 (GLP, Klimisch 1). As result, an in vitro eye irritation score of 10.16 was obtained. Thus, the substance falls into the range of >3- <55 for which no prediction is possible. However, the histopathological examination revealed indications for irritation, consisting of slight multifocal apoptosis/necrosis associated with multifocal vacuolation in the epithelium of all specimen. The occurrence of these effects, which point to a low depth of injury, make it likely that 3,4- epoxycyclohexylmethyl methacrylate should be classified as eye irritant (GHS category 2). As no in vitro test has been validated for the identification of GHS category 2 eye irritants so far, further in vitro testing could not contribute to a better prediction and was therefore omitted at this stage.

Reference:
Scott, 2010. Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alépée, N., Bufo, M., Clothier, R., Facchini, D., Faller, C., Guest, R., Harbell, J., Hartung, T., Kamp, H., Le Varlet, B., Meloni, M., McNamee, P., Osborne, R., Pape, W., Pfannenbecker, U., Prinsen, M., Seaman, C., Spielman, H., Stokes, W., Trouba, K., Van den Berghe, C., Van Goethem, F., Vassallo, M., Vinardell, P., and Zuang, V. (2010). A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom- Up and Top- Down approaches. Toxicol. in Vitro 24:1- 9.


Effects on skin irritation/corrosion: irritating

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

The available data suggest a skin and eye irritation potential. Therefore, 3,4-epoxycyclohexylmethyl methacrylate has to be classified accordingly with "Skin Irrit. 2, H315; Eye Irrit. 2, H319 under the CLP regulation (Regulation (EC) 1272/2008).