Glycine, N,N'-1,2-ethanediylbis-, reaction products with formaldehyde, iron chloride (FeCl3) and phenol, potassium salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

potassium 2-({2-[(carboxymethyl)[(2-hydroxyphenyl)methyl]amino]ethyl}[(2-hydroxyphenyl)methyl]amino)acetic acid iron potassium hydride chloride

EC Number:

938-828-8

Cas Number:

1463474-95-4

Molecular formula:

Not applicable for the reaction product (UVCB)

IUPAC Name:

potassium 2-({2-[(carboxymethyl)[(2-hydroxyphenyl)methyl]amino]ethyl}[(2-hydroxyphenyl)methyl]amino)acetic acid iron potassium hydride chloride

Test material form:

other: microgranules

Details on test material:

Description: dark red - brown microgranules
Batch: CFC-11140 (F501236001-8)
Purity/Composition: 97.2%
Test substance storage: at room temperature in the dark
Stability under storage conditions: stable
Expiry date: 2 October 2015
Test substance handling: no specific handling required

No correction was made for the purity/composition of the test compound.

In vitro test system

Test system:

human skin model

Details on test system:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 13-EKIN-029 and 13-EKIN-034 ).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Amount/concentration applied:

The solid test substance (10.7-12.2 mg) was applied directly on top of the skin tissue.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

109

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

HBED-Fe (UVCB) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that HBED-Fe (UVCB) did not interact with MTT.
The mean absorption at 570 nm measured after treatment with HBED-Fe and controls are presented below Table 1.
Table 2 shows (between brackets) the mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 80%.
The positive control had a mean cell viability of 23% after 15 minutes exposure. The OD570 of two out of three negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated with the substance or positive control was less than 4%. The standard deviation value of the percentage viability of three tissues treated with the negative control was 31%, caused by one out of three tissues. Although the SD was outside the acceptability criteria of the assay, a clear negative result was obtained by the test substance. However, since not all criteria were met, the assay was repeated at request of the sponsor.
In the repeat experiment, the relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 109% (Table 2). Since the mean relative tissue viability for HBED-Fe (UVCB) was above 50% after 15 minutes treatment HBED-Fe (UVCB) is considered to be non-irritant. The positive control had a mean cell viability of 3% after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly (Table 1).

Any other information on results incl. tables

Table1            Mean absorption in the in vitro skin irritation test with HBED-Fe (UVCB) - 2nd experiment

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.180	1.134	1.050	1.121	±	0.066
HBED-Fe (UVCB)	1.239	1.252	1.183	1.224	±	0.036
Positive control	0.046	0.042	0.028	0.039	±	0.010

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.045). Isopropanol was used to measure the background absorption.

 

In the first experiment the mean values +/- SD were as follows:

- Negative control: 1.472 +/- 0.454

- HBED-Fe (UVCB): 1.174 +/- 0.085

- Positive control: 0.408 +/- 0.057

Table2            Mean tissue viability in thein vitroskin irritation test with HBED-Fe (UVCB) - 2nd experiment

	Mean tissue viability as % of control
Negative control	100 (100)
HBED-Fe (UVCB)	109 (80)
Positive control	3 (23)

 Between brackets the results of the first experiment are given.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritating in an in vitro test (OECD 439).

Executive summary:

This report describes the ability of HBED-Fe (UVCB) to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of HBED-Fe (UVCB) was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch CFC 11140 (F501236001-8) of HBED-Fe (UVCB) were dark red-brown microgranules with a purity of 97.2%. Skin tissue was moistened with 5 μl of Milli-Q water and 10.7 to 12.2 mg of HBED-Fe (UVCB) was applied on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 80%. The positive control had a mean cell viability of 23% after 15 minutes exposure. The OD570 (optical density at 570 nm) of two out of three negative control tissues was within the laboratory historical control data range The standard deviation value of the percentage viability of three tissues treated with the substance or positive control was less than 4%. The standard deviation value of the percentage viability of three tissues treated with the negative control was 31%, caused by one out of three tissues. Although the SD was outside the acceptability criteria of the assay, a clear negative result was obtained by the test substance. However, since not all criteria were met, the assay was repeated at request of the sponsor. In the repeat experiment, the relative mean tissue viability obtained after 15 minutes treatment with HBED-Fe (UVCB) compared to the negative control tissues was 109%. Since the mean relative tissue viability for HBED-Fe (UVCB) was above 50% after 15 minutes treatment HBED-Fe (UVCB) is considered to be non-irritant. The positive control had a mean cell viability of 3% after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly. Finally, it is concluded that this test is valid and that HBED-Fe (UVCB) is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.