Heptanal, oligomeric reaction products with aniline

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 April 2016 to 08 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Heptanal, oligomeric reaction products with aniline

EC Number:

500-007-3

EC Name:

Heptanal, oligomeric reaction products with aniline

Cas Number:

9003-50-3

Molecular formula:

(C7H14O)n.(C6H7N)n

IUPAC Name:

Heptanal, oligomeric reaction products with aniline

Test material form:

liquid

Details on test material:

Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Normal human-derived epidermal keratinocytes

Source strain:

other: EpiDerm Skin Model (EPI-200, Lot no.: 23297 kit U and T)

Details on animal used as source of test system:

EpiDerm Skin Model (EPI-200, Lot no.: 23297 kit U and T.
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
Source: MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Cell culture
Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

Freeze-killed tissues
(EPI-200, Lot no.: 23220 kit P, 23240 kit DD, 23297 kit N and 23610 kit M and N)
Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C).
The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 69 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50μl

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2 tissues per test group (3 minutes exposure, 1 hour exposure, negative control, positive control).

Test animals

Species:

other: Human

Strain:

other: EpiDerm Skin Model (EPI-200, Lot no.: 23297 kit U and T)

Details on test animals or test system and environmental conditions:

Test system
EpiDerm Skin Model (EPI-200, Lot no.: 23297 kit U and T)
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Rationale
Recommended test system in international guidelines (OECD and EC).

Source: MatTek Corporation, Ashland MA, U.S.A.

Cell culture Tissues
On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

Freeze-killed tissues (EPI-200, Lot no.: 23220 kit P, 23240 kit DD, 23297 kit N and 23610 kit M and N,)
Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues.

DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 69 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to 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:

open

Preparation of test site:

other: See Details on Study Design

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50μl

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

Negative control:
Milli-Q water (Millipore Corp., Bedford, Mass., USA).
 
Positive control:
Potassium hydroxide (KOH; Merck, Darmstadt, Germany), an 8.0 normal solution was prepared.
 
Test item preparation
No correction was made for the purity/composition of the test compound.
The liquid test item was applied undiluted (50 μl) directly on top of the tissue. Hepteen Base® was spread to match the size of the tissue.
 
Study design
Test for the interference of the test item with the MTT endpoint
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test item
Hepteen Base® was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μl of Hepteen Base® or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.

Test for reduction of MTT by the test item
Hepteen Base® was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of Hepteen Base® was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.
Since the test item reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed non treated tissues were used for the cytotoxicity evaluation with MTT. At the end of the exposure time a blue / purple colour change was observed.

Application/Treatment of the test item
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium is just beneath the tissue (see fig 1). The plates were incubated for approximately 2 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before Hepteen Base® was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Hepteen Base® and two for a 1-hour exposure. Fifty μl of the undiluted test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. In addition for the 3 minute and 1 hour exposure two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.
 
Cell viability measurement
The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Evaluation
Acceptability of the assay
Thein vitroskin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 3-minute exposure to the positive control should be ≤ 30%.
c) In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.
 
Data evaluation and statistical procedures
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in thein vitroskin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
 
ELECTRONIC SYSTEMS FOR DATA ACQUISITION
The following electronic systems were used for data acquisition:
-REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA).
- Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Not corrosive

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Not corrosive

Other effects / acceptance of results:

Hepteen Base® was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that Hepteen Base® did interact with the MTT endpoint.
In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by Hepteen Base® was 10% and 21% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Hepteen Base® compared to the negative control tissues was 68%. Because the mean relative tissue viability for Hepteen Base® was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment Hepteen Base® is considered to be not corrosive.

Any other information on results incl. tables

Mean absorption in thein vitroskin corrosion test with Hepteen Base®

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD		A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	2.403	2.173	2.288	±	0.162	2.105	2.188	2.146	±	0.059
Hepteen Base®(1)	1.792	1.319	1.556	±	0.334	1.482	1.436	1.459	±	0.032
Positive control	0.168	0.204	0.186	±	0.026	0.191	0.194	0.192	±	0.002

Duplicate exposures are indicated by A and B.

(1)The values are corrected for the non-specific MTT reaction (10% and 21% at the 3 minute and 1 hour treatment, respectively).

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

Mean tissue viability in thein vitroskin corrosion test with Hepteen Base®

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Hepteen Base®	68	68
Positive control	8	9

 

	Coefficient of Variation between tissue replicates3 minute	1 hour
Negative control	9.6	3.8
Hepteen Base®	26.4	3.1
Positive control	17.8	1.8

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

INDIVIDUAL OD MEASUREMENTS AT 570 NM

	3-minute application (OD570) A B		1-hour application (OD570) A B
Negative control
OD570measurement 1	2.4630	2.2175	2.1832	2.2288
OD570measurement 2	2.4341	2.2039	2.1094	2.2138
OD570measurement 3	2.4339	2.2212	2.1452	2.2451
Hepteen Base®
OD570measurement 1	2.0879	1.5983	1.9673	1.9398
OD570measurement 2	2.0513	1.5937	1.9700	1.8808
OD570measurement 3	2.0573	1.5861	1.9604	1.9399
Hepteen Base® on freeze killed tissue
OD570measurement 1	0.4125	0.4528	0.6840	0.6014
OD570measurement 2	0.4032	0.4449	0.6631	0.5838
OD570measurement 3	0.4085	0.4360	0.6709	0.5910
Negative control - treated freeze killed tissue
OD570measurement 1	0.1929	0.1946	0.1682	0.2111
OD570measurement 2	0.1947	0.1957	0.1682	0.2118
OD570measurement 3	0.1933	0.1934	0.1681	0.2109
Positive control
OD570measurement 1	0.2130	0.2436	0.2319	0.2339
OD570measurement 2	0.2047	0.2481	0.2329	0.2347
OD570measurement 3	0.2094	0.2444	0.2305	0.2370

OD = Optical density

Duplicate exposures are indicated by A and B.

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.324 – 2.615	1.361 – 2.352	0.172 – 0.56	0.057 – 0.277	6 – 22	3 – 12
Mean	1.86	1.86	0.18	0.13	10.67	7.17
SD	0.24	0.22	0.10	0.05	3.9	2.36
n	65	67	64	61	30	30

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of December 2012 to December 2015.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that Hepteen Base® is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with Hepteen Base® using a human skin model.

 

This report describes the ability of Hepteen Base® to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of Hepteen Base® was tested through topical application for 3 minutes and 1 hour.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 

Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid with a purity of 99.7%. Hepteen Base® was applied undiluted (50 μl) directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 8% after 3 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was ≤ 27%, indicating that the test system functioned properly.

 

Hepteen Base® did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by Hepteen Base® was 10% and 21% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Hepteen Base® compared to the negative control tissues was 68%. Because the mean relative tissue viability for Hepteen Base® was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment Hepteen Base® is considered to be not corrosive.

 

In conclusion Hepteen Base® is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.