Formaldehyde, reaction products with ethylenediamine

Endpoint summary

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:

July 11- 16, 2012

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Qualifier:

according to guideline

Guideline:

other: L’Oréal Standard Operating Procedure: “EpiSkin™ Skin Irritation Test Method“; - ECVAM Skin Irritation Validation Study - Validation of the EpiSkin™ Test Method15 min - 42 hours for the Prediction of Acute Skin Irritation of Chemicals. Version 1.8, 02-2009

Qualifier:

according to guideline

Guideline:

other: ECVAM Performance Standards for in vitro Skin Irritation Test Methods based on Reconstructed human Epidermis (RhE). Updated Version 24-Aug-2009

Qualifier:

according to guideline

Guideline:

other: ISO 10993-10, Annex D: 2010, “Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization”

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The test will be carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum

Species:

other: human skin model

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 92

Vehicle controls validity:

valid

Negative controls validity:

not specified

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM™ (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study Formaldehyde, reaction product with ethylenediamine was applied topically to the EPISKIN-SM™ tissue for 15 min followed by a 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (92%) 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 (92%) 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% (7%). The maximum standard deviation of viability of replicate tissues of all dose groups was < 18% (3.4% - 9.8%)

Interpretation of results:

not irritating

Remarks:

Migrated information

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance
with UN GHS and EU CLP “No Category”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

May 30, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Principles of method if other than guideline:

The Bovine Corneal Opacity and Permeability (BCOP) test method is an ex vivo test method that can be used to classify substances as “ocular corrosives and severe irritants”. The BCOP is recommended for use as part of a tiered-testing strategy for regulatory classification and labelling within a specific applicability domain. Test substances can be classified as ocular corrosives or severe irritants without further testing in rabbits.
To ensure test method integrity the data of the BCOP assay (Bovine Corneal Opacity and Permeability Assay), reference substances are tested in parallel to the test item.

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Irritation parameter:

in vitro irritation score

Value:

ca. 29

Negative controls validity:

valid

Positive controls validity:

valid

The eye irritancy potential of Formaldehyde, reaction product with ethylenediamine was investigated in the bovine corneal opacity and permeability assay.

The following mean in vitro irritation score was calculated: 29.32

Therefore the test item was classified as moderate irritant.

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.

OPACITY

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	3	7	4
2		3	3	0
3		3	4	1
MV		3.0	4.67	1.67
4	Positive Control	4	101	97	95.33
5		4	59	55	53.33
6		4	69	65	63.33
MV		4.0	76.33	72.33	70.67
7	Test Item	2	35	33	31.33
8		2	32	30	28.33
9		2	32	30	28.33
MV		2.0	33.00	31.00	29.33

PERMEABILITY

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.038
2		0.024
3		0.031
MV		0.031
4	Positive Control	0.370	0.039
5		0.183	0.152
6		0.328	0.297
MV		0.294	0.263
7	Test Item	0.037	0.006
8		0.004	-0.027
9		0.049	0.018
MV		0.030	-0.001

IN VITRO IRRITATION SCORE

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	4.00	0.038
2		0.00	0.024
3		1.00	0.031
MV		1.67	0.031	2.13
4	Positive Control	95.33	0.039
5		53.33	0.152
6		63.33	0.297
MV		70.67	0.263	74.61
7	Test Item	31.33	0.006
8		28.33	-0.027
9		28.33	0.018
MV		29.33	-0.001	29.32

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Formaldehyde, reaction product with ethylenediamine is classified as Eye Irrit 2

Executive summary:

The test was considered valid to confirm classification as Eye Irrit 2 being needed. No further animal testing is justified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The results of the in vitro tests performed with formaldehyde, reaction products with ethylendiamine show neither corrosive nor irritation properties related to the test item. The ex vivo study performed to evaluate the irritation potential of the tet substance to the eyes, showed a moderate irritating potential.

Justification for selection of skin irritation / corrosion endpoint:

As in the skin corrosion study formaldehyde, reaction products with ethylendiamine resulted non corrosive, a skin irritation study has been carried out. Also this study gave negative results and therefore it has been choosen as endpoint selection study.

Effects on eye irritation: moderately irritating

Justification for classification or non-classification

According to the results of the available studies, formaldehyde, reaction products with ethylendiamine is classified as follows, according to Regulation 1272/2008/EC:

Eye Irrit. 2, H319.