Heptanal, oligomeric reaction products with aniline

Endpoint summary

Description of key information

In vitro Skin Corrosion & In vitro Skin Irritation

Key values were determined in a GLP accredited laboratory using a human skin models and performed in accordance with OECD Guideline no. 431, 439 and E.U B.40, B.46 test guidelines.

 

HEPTEEN BASE® was not corrosive under the conditions of the in vitro skin corrosion/irritation tests under experimental conditions.

In vivo Skin Irritation/corrosion

Key values were determined in a GLP accredited laboratory using a human skin models and performed in accordance with OECD Guideline no. 404, E.U B.4, USEPA 712-C-98-196, and JMAFF 8147 test guidelines.

 

HEPTEEN BASE® was considered to be a skin irritant under the conditions of the in vivo skin irritation/corrosion experimental conditions described in the report.

In vitro Eye Damage/Irritation

Key values were obtained in a GLP accredited laboratory using the Bovine Corneal Opacity and Permeability Test in accordance with OECD test guideline 437.

HEPTEEN BASE® did not induce ocular corrosion/irritation under the conditions of the experimental conditions.

In vivo Eye Irritation/corrosion

Key values were determined in a GLP accredited laboratory using a human skin models and performed in accordance with OECD Guideline no. 405, E.U B.5, USEPA 712-C-98-195, and JMAFF 8147 test guidelines.

 

HEPTEEN BASE® was considered to be an eye irritant under the conditions of the in vivo eye irritation/corrosion experimental conditions.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 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).

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.

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.

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

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.

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.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2016 to 17 May 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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

ST.DEV of viability of test item treated tissues = 24%, which is above the acceptance criteria. All individual viabilities were >50%, clearly negative and did not influence the outcome. Study integrity not adversely affected by deviation.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

ST.DEV of viability of test item treated tissues = 24%, which is above the acceptance criteria. All individual viabilities were >50%, clearly negative and did not influence the outcome. Study integrity not adversely affected by deviation.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: adult human derived epidermal keratinocytes

Cell source:

other: Not specified

Source strain:

other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-019,

Details on animal used as source of test system:

Source
SkinEthic Laboratories, Lyon, France.

Preparation and pre-incubation
Tissues
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for approximately 24 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 item 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 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 – 37.0°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.

Justification for test system used:

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).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-019).
This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 μl

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

2 hours

Number of replicates:

2 tissues per group (test substance, positive control, negative control).

Species:

other: Human

Strain:

other: EPISKIN Small Model (EPISKIN-SMTM)

Details on test animals or test system and environmental conditions:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-019) This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

All incubations, with the exception of the test item 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 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 – 37.0°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.

Type of coverage:

open

Preparation of test site:

other: See Study Design

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

25 μl

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on 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.
Hepteen Base® was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 512983). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Hepteen Base® did not interfere with the MTT endpoint.

Application/Treatment of the test item
The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item 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 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. 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-solution (0.3 mg/ml in PBS). 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 approximately 71 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 item was classified according to remaining cell viability following exposure of the test item.

Evaluation
Acceptability of the assay
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Data evaluation and statistical procedures
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

80

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Hepteen Base® compared to the negative control tissues was 80%. Since the mean relative tissue viability for Hepteen Base® was above 50% Hepteen Base® is
considered to be non-irritant.

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12%. 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 13% for the negative and positive control, indicating that the test system functioned properly. For the test item treated replicates the standard deviation was less than 24%, however since all three individual viabilities were >50%, this does not influence the study outcome.

Mean absorption in thein vitroskin irritation test with Hepteen Base®

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	0.755	0.924	0.968	0.882	± 0.112
Hepteen Base®	0.947	0.630	0.551	0.709	± 0.210
Positive control	0.115	0.136	0.069	0.107	± 0.035

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 (0.041). Isopropanol was used to measure the background absorption.

 

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

	Mean tissue viability (percentage of control)
Negative control	100
Hepteen Base®	80
Positive control	12

 

Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control
OD570measurement 1	0.8261	0.9820	1.0665
OD570measurement 2	0.7665	0.9482	0.9509
Hepteen Base®
OD570measurement 1	0.9863	0.6712	0.6022
OD570measurement 2	0.9903	0.6706	0.5820
Positive control
OD570measurement 1	0.1570	0.1895	0.1112
OD570measurement 2	0.1562	0.1653	0.1086

OD = Optical density

Triplicate exposures are indicated by A, B and C.

 

Historical control data forin vitroskin irritation studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	0.676 – 1.336	0.028 – 0.408	2.46 – 42.99
Mean	1.06	0.16	14.78
SD	0.14	0.11	9.29
n	115	115	115

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.

Interpretation of results:

GHS criteria not met

Conclusions:

To conclude, this test is valid and that Hepteen Base® is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

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

 

This report describes the ability of Hepteen Base® to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of Hepteen Base® 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 LT5C30Y170 of the test item was a clear amber liquid with a purity of 99.7%. The test item was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 80%. Since the mean relative tissue viability for Hepteen Base® was above 50% after 15 ± 0.5 minutes treatment Hepteen Base® is considered to be non-irritant.

 

The positive control had a mean cell viability of 12% after 15 ± 0.5 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 13% for the negative and positive control, indicating that the test system functioned properly. For the test item treated replicates the standard deviation was less than 24%, however since all three individual viabilities were >50%, this does not influence the study outcome.

 

To conclude this test is valid and that Hepteen Base® is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

 

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June 2016 to 02 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted in accordance with OECD, EU, US-EPA and JMAFF test guidelines in an accredited GLP laboratory.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015
1. Deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sentinel animal was a female, the other two animals were males.
Evaluation: The sexes are considered similar in sensitivity. The results of this study confirm this.
The study integrity was not adversely affected by the deviation.

Deviations:

yes

Remarks:

see remarks

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

May 2008, including most recent amendments.

1. Deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sentinel animal was a female, the other two animals were males.
Evaluation: The sexes are considered similar in sensitivity. The results of this study
confirm this.
The study integrity was not adversely affected by the deviation.

Deviations:

yes

Remarks:

see remarks

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

EPA 712-C-98-196, August 1998.

1. Deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sentinel animal was a female, the other two animals were males.
Evaluation: The sexes are considered similar in sensitivity. The results of this study
confirm this.
The study integrity was not adversely affected by the deviation.

Deviations:

yes

Remarks:

see remarks

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147

Version / remarks:

November 2000; including the most recent partial revisions.

1. Deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. The sentinel animal was a female, the other two animals were males.
Evaluation: The sexes are considered similar in sensitivity. The results of this study confirm this.
The study integrity was not adversely affected by the deviation.

Deviations:

yes

Remarks:

see remarks

GLP compliance:

yes

Specific details on test material used for the study:

pH (1% in water, indicative range): 8.0 – 7.8 (determined by Charles River Den Bosch)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Species: Albino rabbit, New Zealand White, (SPF-Quality).
Recognized by international guidelines as the recommended test system (e.g. EC, OECD)
Source: Charles River France, L’Arbresle, France
Number of animals: 1 Female and 2 males.
Age and body weight: At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg.
Identification: Earmark.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the skin to be treated was intact and free from any abnormality.

Animal Husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.5mL

Duration of treatment / exposure:

Exposure time: 4 hours

Observation period:

14 days

Number of animals:

Total of 3 animals

Details on study design:

Test Item Preparation
The test item was applied undiluted as delivered by the Sponsor.
No corrections were made for the purity/composition of the test item, since the guidelines require a fixed amount that has to be applied.

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later, after considering the degree of skin irritation observed in the first animal.

Treatment
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimetres (10x15 cm). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations.

Each animal was treated by dermal application of 0.5 mL of the test item. The test item was applied to the skin of one flank, using a metalline patch# of 2x3 cm. The patch was mounted on Micropore tape#, which was wrapped around the abdomen and secured with Coban elastic bandage#.

Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water.
#. Suppliers: Lohmann & Rauscher B.V., Almere, The Netherlands (Metalline) and 3M, St. Paul, Minnesota, U.S.A. (Micropore and Coban).

After the final observation, the first animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The other two animals were removed from the study alive.

Observations
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body: Weight Day of treatment (prior to application) and on the day of the final observation.
Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 and 14 days after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.

The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
No erythema = 0
Very slight erythema (barely perceptible) = 1
Well-defined erythema = 2
Moderate to severe erythema = 3
Severe erythema (beef redness) * = 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
No oedema = 0
Very slight oedema (barely perceptible) = 1
Slight oedema (edges of area well-defined by definite raising) = 2
Moderate oedema (raised approximately 1 millimeter) = 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) = 4

Necropsy No necropsy was performed according to study plan.

No histopathology was performed.

Irritation parameter:

erythema score

Remarks:

Sentinel

Basis:

animal: 111

Time point:

other: Mean 24, 48 and 72 hrs

Score:

2

Max. score:

4

Reversibility:

not fully reversible within:

Remarks:

14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Remarks:

Sentinel

Basis:

animal: 111

Time point:

other: Mean 24, 48 and 72 hrs

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: 125

Time point:

other: Mean 24, 48, 72 hrs

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: 125

Time point:

other: Mean 24, 48, 72 hrs

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within:

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: 126

Time point:

other: Mean 24, 48, 72 hrs

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: 126

Time point:

other: Mean 24, 48, 72 hrs

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Irritation
Four hours exposure to 0.5 mL of Hepteen Base® resulted in very slight to severe erythema and very slight to moderate oedema in the treated skin areas of the three rabbits. This resulted in reduced flexibility of the skin, scaliness, fissuring of the skin, scabs (formation), white coloration of the skin (sign of necrosis) and/or bald skin at 7 and/or 14 days after instillation.

The erythema and oedema resolved within 14 days after exposure in two animals, the other animal showed erythema at 14 days after exposure. Scaliness was visible at 14 days after exposure in all three animals. Additionally, one of the animals showed scabs and one of the animals showed bald skin at 14 days after exposure.
The sentinel animal showed erythema outside of the application area at 7 and 14 days after exposure.

Corrosion
There was no evidence of a corrosive effect on the skin.

Other effects:

Coloration / Remnants
No staining of the treated skin by the test item was observed and no test item remnants were seen.

Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual Skin Irritation Scores

Animal	111^1			125			126
Time after exposure	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments	Erythema (0-4)	Oedema (0-4)	Comments
1 hour	0	0	-	1	1	-	1	1	-
24 hours	2	3	-	2	2	-	2	2	-
48 hours	2	3	-	2	2	-	2	2	-
72 hours	2	3	-	2	3	-	2	2	R
7 days	3	3	F, o	3	3	F, g	4	3	K, g, d, f
14 days	1	0	L, o	0	0	E, l	0	0	H, l

1 Sentinel.

Comments:

d. Scab formation

e. Scabs (deep red/ brown)

f. Reduced flexibility of the skin

g. Fissuring of the skin

h. Bald skin

k. White coloration of the skin (sign of necrosis)

l. Scaliness

r. Scattered erythema

o. Erythema outside of application area

Mean irritation scores

Animal	Mean 24, 48, 72 hrs
	erythem	oedema
111	2.0	3.0
125	2.0	2.3
126	2.0	2.0

 

Animal specifications

Animal	Sex	Age at start (weeks)	Body weight (g)
			Prior to application	After the final observation
111	Female	21	3964	4111
125	Male	12	2733	3070
127	Make	12	2526	2953

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the persistence of the skin reactions:
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Hepteen Base® should be classified as: skin irritant (Category 2).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base® should be classified as should be classified as Irritant (Category 2) and labeled as H315: Causes skin irritation.

Executive summary:

Primary skin irritation/corrosion study with Hepteen Base® in the rabbit (4-hour semiocclusive application).

 

The study was carried out based on the guidelines described in:

OECD No.404, "Acute Dermal Irritation/Corrosion" (2015)

EC, No 440/2008; B4: "Acute Toxicity: Dermal Irritation/Corrosion".

US EPA, OPPTS 870.2500 (1998), Acute Dermal Irritation.

JMAFF Guidelines (2000), including the most recent revisions.

 

Three rabbits were exposed to 0.5 mL of Hepteen Base®, by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 and 14 days after exposure.

 

Four hours exposure to 0.5 mL of Hepteen Base® resulted in very slight to severe erythema and very slight to moderate oedema in the treated skin areas of the three rabbits. This resulted in reduced flexibility of the skin, scaliness, fissuring of the skin, scabs (formation), white colouration of the skin and/or bald skin at 7 and/or 14 days after instillation.

 

The erythema and oedema resolved within 14 days after exposure in two animals, the other animal showed erythema at 14 days after exposure. Scaliness was visible at 14 days after exposure in all three animals. Additionally, one of the animals showed scabs and one of the animals showed bald skin at 14 days after exposure.

 

Based on the persistence of the skin reactions:

-according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Hepteen Base® should be classified as: skin irritant (Category 2).

-according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base® should be classified as should be classified as Irritant (Category 2) and labeled as H315: Causes skin irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2016 to 11 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted according to OECD test guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter but within 4 hours.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter but within 4 hours.

Transport: Eyes were collected and transported in physiological saline under cooled conditions (in a cooling box with iced elements).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent negative control

Amount / concentration applied:

750 μl

Duration of treatment / exposure:

10 ± 1 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3 corneas per test group (test substance, positive control, negative control)

Details on study design:

Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
In the first test an inappropriate response of the negative control was observed (not reported), therefore a repeat test was performed. The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Opacity measurement
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = (((I0/I)-0.9894)/ 0.0251)
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg
Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

other: Mean tissue viability (%)

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No other effects noted.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. Therefore the test system functioned properly

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity1	Mean Permeability1	Mean In vitro Irritation Score1, 2
Negative control	-1.2	0.002	-1.1
Positive control (Ethanol)	19.7	1.039	35.3
Hepteen Base®	1.1	0.004	1.2

 

1 Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final opacity^1	Negative control corrected Final Opacity^2	Mean final opacity
Negative control	2.9	0.8	2.1		-1.2
	4.9	4.7	-0.1
	3.9	2.7	-1.2
Positive control	3.7	22.4	20.6	20.6	19.7
	3.5	24.0	20.4	20.4
	4.2	22.3	18.1	18.1
Hepteen base ®	5.0	5.4	0.4	0.4	1.1
	4.1	5.7	1.6	1.6
	4.2	5.5	1.2	1.2

1Final Opacity = Opacity after treatment – Opacity before treatment.

2Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.3

3In case the mean final opacity of the negative control is below zero, no correction will be made.

4Calculations are made without rounding off.

Permeability score individual values (uncorrected)

 

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.001	0.002	0.002	0.002	0.002	0.002
	1	0.002	0.002	0.002	0.002	0.002
	1	0.004	0.003	0.003	0.003	0.003
Positive control	1	0.880	0.873	0.873	0.880	0.880
	1	0.875	0.871	0.871	0.873	0.873
	1	1.380	1.371	1.371	1.373	1.373
Hepteen base ®	1	0.009	0.008	0.008	0.008	0.008
	1	0.003	0.004	0.004	0.003	0.003
	1	0.007	0.008	0.008	0.008	0.008

 

1Calculations are made without rounding off.

Permeability score individual values (corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD490
Positive control	1	0.878	0.84	0.871	0.877	0.877	1.039
	1	0.873	0.870	0.869	0.870	0.870
	1	1.378	0.366	1.369	1.371	1.371
Hepteen Base®	1	0.007	0.006	0.006	0.006	0.006	0.004
	1	0.001	0.001	0.002	0.001	0.001
	1	0.005	0.001	0.006	0.005	0.005

1OD490values corrected for the mean final negative control permeability (0.002).

2Calculations are made without rounding off.

In Vitroirritancy score

Treatment	Final Opacity^2	Final OD490^2	In vitro irritancy score^1
Negative control	-2.1	0.002	-2.1
	-0.1	0.002	-0.1
	-1.2	0.003	-1.2
Positive control	20.6	0.877	33.8
	20.4	0.870	33.5
	18.1	1.371	38.7
Hepteen base ®	0.4	0.006	0.5
	1.6	0.001	1.6
	1.2	0.005	1.3

1In vitroirritancy score (IVIS) = opacity value + (15 x OD490value).

2Positive control and test item are corrected for the negative control.

Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9-3.0	-0.016-0.019	-2.8-3.0	35.9-63.9
Mean	0.22	0.00	0.30	50.74
SD	1.12	0.01	1.18	10.34The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 1.2 after 10 minutes of treatment. Since Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
n	39	34	36	12

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 February 2015 to January 2016.

Interpretation of results:

GHS criteria not met

Conclusions:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.2 after 10 minutes of treatment.

Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye damage of Hepteen Base® was tested through topical application on bovine eyes for 10 minutes.The study procedures were based on the most recent OECD guideline.

 

Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid a purity of 97.3%. The test item was applied as it is (750 μl) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a mean in vitroirritancy score of 1.2 after 10 minutes of treatment.

Since Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2016 to 11 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted in accordance with OECD, EU, EPA and JMAFF guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012.

Deviations:

yes

Remarks:

Deviations in daily max relative humidity

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

May 2008, including most recentamendments.

Deviations:

yes

Remarks:

Deviations in daily max relative humidity

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

August 1998

Deviations:

yes

Remarks:

May 2008, including most recentamendments.

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan,Notification No 8147

Version / remarks:

November 2000; including the most recent partial revisions.

Deviations:

yes

Remarks:

Deviations in daily max relative humidity

GLP compliance:

yes

Specific details on test material used for the study:

pH (1% in water, indicative range) 8.0 – 7.8 (determined by Charles River Den Bosch)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Albino rabbit, New Zealand White, (SPF-Quality).
Source: Charles River France, L’Arbresle, France
Number of animals 2 Males and 1 Female.
Age and body weight: At start of dosing, the animals were between 12 and 21 weeks old and body weights were at least 1.5 kg.
Identification: Earmark.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the eyes were free from any abnormality.
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod was between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet
Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period.

Water
Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

21 days after dosing

Number of animals or in vitro replicates:

A total of 3 animals (2 male and 1 female), one of which was used as a sentinel

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner four weeks later, after considering the degree of eye irritation observed in the first animal.

Irritation parameter:

cornea opacity score

Basis:

animal: 91

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 101

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 102

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 91

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 101

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal: 102

Time point:

24/48/72 h

Score:

0.3

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal: 91

Time point:

24/48/72 h

Score:

1.3

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal: 101

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal: 102

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal: 91

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal: 101

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal: 102

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible

Other effects:

Irritation
Instillation of 0.1 mL of Hepteen Base® into one eye of each of three rabbits resulted in irritation of the cornea, iris and/or conjunctivae.
The corneal injury, observed in one animal only (no. 102), consisted of opacity and epithelial damage, at 24 hours after instillation only. Iridial irritation was noted for two animals (no. 101 and 102), at 24 hours after instillation only. The irritation of the conjunctivae consisted of redness, chemosis and discharge in all animals. The irritation had completely resolved within 7 days in one animal (no. 91) and within 21 days in the other two animals (no. 101 and 102).

Corrosion
There was no evidence of ocular corrosion.

Coloration / Remnants
No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen.

Toxicity / Mortality
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual Eye Irritation Scores

Animal	Time after dosing	Cornea			Iris	Conjunctivae			Comments
		Opacity (0-4)	Area (0-4)	Fluor area (%)2	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
911	1 hour	0	0		0	2	1	1	-
	24 hours	0	0	0	0	2	2	2	-
	48 hours	0	0		0	1	1	1	-
	72 hours	0	0		0	1	0	1	-
	7 days	0	0		0	0	0	0	-
101	1 hour	0	0		0	2	2	2	-
	24 hours	0	0	0	1	2	2	3	-
	48 hours	0	0		0	2	1	1	-
	72 hours	0	0		0	2	1	1	-
	7 days	0	0		0	1	0	0	-
	14 days	0	0		0	1	0	0	-
	21 days	0	0		0	0	0	0	-
102	1 hour	0	0		0	2	2	2	-
	24 hours	0	1	25	1	2	2	2	g
	48 hours	0	0		0	2	1	1	-
	72 hours	0	0	0	0	2	1	1	-
	7 days	0	0		0	1	0	0	-
	14 days	0	0		0	1	0	0	-
	21 days	0	0		0	0	0	0	-

 

1Sentinel

2Green staining after fluorescein treatment (percentage of total corneal area)

Comments: g Slight dulling of the normal luster of the cornea.

 

 

Animal	sex	Age at start (weeks)	Body weight
			Prior to application	At termination
91	Female	21	4445	4618
101	male	12	2244	2733
102	male	12	2485	2811

 

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

Based on these results:
According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Hepteen Base® should be classified as: irritating to eyes (Category 2A).

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base® should be classified as Irritating to eyes (Category 2) and labelled as H319: Causes serious eye irritation.

Executive summary:

Acute eye irritation/corrosion study with Hepteen Base® in the rabbit.

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"

EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation" JMAFF Guidelines (2000), including the most recent revisions.

 

Single samples of 0.1 mL of Hepteen Base® were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days and/or 14 and 21 days after instillation.

 

Instillation of the test item resulted in irritation of the cornea, iris and/or conjunctivae.

 

The corneal injury, observed in one animal only, consisted of opacity and epithelial damage, at 24 hours after instillation only. Iridial irritation was noted for two animals, at 24 hours after instillation only. The irritation of the conjunctivae consisted of redness, chemosis and discharge in all animals. The irritation had completely resolved within 7 days in one animal and within 21 days in the other two animals.

 

Based on these results:

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Hepteen Base® should be classified as: irritating to eyes (Category 2A).

 

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base® should be classified as Irritating to eyes (Category 2) and labelled as H319: Causes serious eye irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In Vitro Skin Corrosion

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 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 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.

 

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.

 

In Vitro Skin Irritation

Batch LT5C30Y170 of the test item was a clear amber liquid with a purity of 99.7%. The test item was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 80%. Since the mean relative tissue viability for Hepteen Base® was above 50% after 15 ± 0.5 minutes treatment Hepteen Base® is considered to be non-irritant.

 

The positive control had a mean cell viability of 12% after 15 ± 0.5 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 13% for the negative and positive control, indicating that the test system functioned properly. For the test item treated replicates the standard deviation was less than 24%, however since all three individual viabilities were >50%, this does not influence the study outcome (study plan deviation 1).

 

It is concluded that this test is valid and that Hepteen Base® is non-irritant in the in vitro skin irritation test under the experimental conditions.

 

In vivo Skin Corrosion

Three rabbits were exposed to 0.5 mL of Hepteen Base®, by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 and 14 days after exposure.

 

Four hours exposure to 0.5 mL of Hepteen Base® resulted in very slight to severe erythema and very slight to moderate oedema in the treated skin areas of the three rabbits. This resulted in reduced flexibility of the skin, scaliness, fissuring of the skin, scabs (formation), white colouration of the skin and/or bald skin at 7 and/or 14 days after instillation.

 

The erythema and oedema resolved within 14 days after exposure in two animals, the other animal showed erythema at 14 days after exposure. Scaliness was visible at 14 days after exposure in all three animals. Additionally, one of the animals showed scabs and one of the animals showed bald skin at 14 days after exposure.

 

It was concluded that this test is valid and that Hepteen Base® is classified as a skin irritant (Category 2) in the in vivo skin irritation test under the experimental conditions

 

In Vitro Eye Damage/Irritation

Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid a purity of 97.3%. The test item was applied as it is (750 μl) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 1.2 after 10 minutes of treatment. Hepteen Base® induced an IVIS ≤ 3

 

It is concluded that this test is valid and that Hepteen Base® is not classified as an eye irritant or eye damage in the in vitro eye irritation/damage test under the experimental conditions.

 

In Vivo Eye Damage /Irritation

Single samples of 0.1 mL of Hepteen Base® were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days and/or 14 and 21 days after instillation.

Instillation of the test item resulted in irritation of the cornea, iris and/or conjunctivae.

The corneal injury, observed in one animal only, consisted of opacity and epithelial damage, at 24 hours after instillation only. Iridial irritation was noted for two animals, at 24 hours after instillation only. The irritation of the conjunctivae consisted of redness, chemosis and discharge in all animals. The irritation had completely resolved within 7 days in one animal and within 21 days in the other two animals.

It was concluded that this test is valid and that Hepteen Base® is classified as a eye irritant (Category 2) in the in vivo eye irritation test under the experimental conditions.

 

 

Justification for classification or non-classification

In vitro Skin Corrosion/Irritation

Based on the results HEPTEEN BASE ® does not have to be classified and has no obligatory labelling requirements for skin corrosion/irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and prepartations (Council Directive 67/548/EEC) (including all amendments).

In vivo Skin Corrosion/Irritation

Based on the in vivo test results conducted on HEPTEEN BASE ® has to be classified as Skin Irritant Category 2, H315 and labelled according to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and prepartations (Council Directive 67/548/EEC) (including all amendments).

In vitro Eye Damage/Irritation

Based on the results Hepteen Base does not have to be classified and has no obligatory labelling requirements for eye damage/irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

In vivo Eye Corrosion/Irritation

Based on these results Hepteen Base ® should be classified as irritating to eyes (Category 2A) and as such is obligated to adhere to the labelling requirements according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments).

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Hepteen Base® should be classified and labelled as Irritating to eyes (Category 2) and labelled as H319: Causes serious eye irritation.