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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 30 April may to 09 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD 439 compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

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)
Remarks:
06 May 2013

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-Benzenedicarboxylic acid, compd. with 1,6-hexanediamine (1:1)
EC Number:
700-230-8
Cas Number:
3160-86-9
Molecular formula:
C8-H6-O4.C6-H16-N2
IUPAC Name:
1,4-Benzenedicarboxylic acid, compd. with 1,6-hexanediamine (1:1)
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
human
Strain:
other: skin model
Details on test animals or test system and environmental conditions:
Test system
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-018).
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.

Preparation and preincubation
Tissues
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 26 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium
MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37 ± 1°C
(actual range 36.0 - 36.9°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity occurred that were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: not applicable
Duration of treatment / exposure:
15 minutes
Number of animals:
Not applicable
Details on study design:
Test for reduction of MTT by the test substance:
Terephthalic acid, hexane-1,6-diamine (1:1) was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 11.0 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance:
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (11.06 to 12.06 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µL PBS (negative control) and 3 tissues with 25 µL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Electronic data capture:
Observations/measurements in the study were recorded electronically using the following programme(s):
REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.
Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Tissue viability
Value:
118
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes. (migrated information)

In vivo

Irritant / corrosive response data:
Terephthalic acid, hexane-1,6-diamine (1:1) 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 Terephthalic acid, hexane-1,6-diamine (1:1) did not interact with MTT.
Results shows the mean tissue viability obtained after 15 minutes treatment with Terephthalic acid, hexane-1,6-diamine (1:1) 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 Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues was 118%. Since the mean relative tissue viability for Terephthalic acid, hexane-1,6-diamine (1:1) was above 50% Terephthalic acid, hexane-1,6-diamine (1:1) is considered to be non-irritant.
The positive control had a mean cell viability after 15 minutes exposure of 10%. 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 9%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean absorption in thein vitroskin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1)

 

 

A

(OD570)

B

(OD570)

C

(OD570)

Mean

(OD570)

 

SD

Negative control

0.966

1.118

1.135

1.073

±

0.093

Test substance

1.322

1.214

1.253

1.263

±

0.055

Positive control

0.084

0.108

0.135

0.109

±

0.025

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)

 

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Table 2: Mean tissue viability in thein vitroskin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1):

 

Mean tissue viability (percentage of control)

Negative control

100

Test substance

118

Positive control

10

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)

Individual OD measurements at 570 nm: 

 

A

(OD570)

B

(OD570)

C

(OD570)

Negative control

OD570measurement 1

OD570measurement 2

1.021

0.993

1.216

1.101

1.192

1.160

Test substance

OD570measurement 1

OD570measurement 2

1.390

1.336

1.361

1.149

1.310

1.278

Positive control

OD570measurement 1

OD570measurement 2

0.137

0.113

0.142

0.156

0.174

0.178

OD = Optical density

Triplicate exposures are indicated by A, B and C.

 

Test substance = Terephthalic acid, hexane-1,6-diamine (1:1)

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that this test is valid and that Terephthalic acid, hexane-1,6-diamine (1:1) is non-irritant in the in vitro skin irritation test under the experimental conditions.
Executive summary:

This study (2014) is an in vitro skin irritation test with Terephthalic acid, hexane-1,6-diamine (1:1) using a human skin model, performed according to OECD 439 guideline and under GLP conditions.

Skin tissue was moistened with 5 µL of Milli-Q water and 11.06 to 12.06 mg of Terephthalic acid, hexane-1,6-diamine (1:1) was applied directly 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 Terephthalic acid, hexane-1,6-diamine (1:1) compared to the negative control tissues was 118%. Since the mean relative tissue viability for Terephthalic acid, hexane-1,6-diamine (1:1) was above 50% after 15 minutes treatment Terephthalic acid, hexane-1,6-diamine (1:1) is considered to be non-irritant.

 

The positive control had a mean cell viability of 10% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) 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 9%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Terephthalic acid, hexane-1,6-diamine (1:1) is non-irritant in the in vitro skin irritation test under the experimental conditions.

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