Norethisterone

Endpoint summary

Description of key information

With respect to local irritant effects, a batterie of in vitro tests for skin irritation/corrosion (OECD TG 431 and 439) and damage to the eye (OECD TG 437, HCE, HET-CAM) is available for Norethisterone. Based on these assays no corrosive or irritant potential to the skin and no damage to the eye can be concluded for the substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:

- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Value:

110.21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- DEMONSTRATION OF TECHNICAL PROFICIENCY:

Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Tabular Summary of the results

Sample No.	Test item	OD mean#	StdDev	% Viability
1-3	Negative control NaCl 0.9 %	2.26	0.06	100.00
4-6	Positive control SDS 5 %	0.02	0.00	0.98
7 -9	Norethisteron, gesiebt (D)	2.50	0.13	110.21

# 6 values

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 110 %. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2014

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min- exposure and incubator (37
± 2° C) for 60 min- exposure

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 inserts was determined by duplicate per insert = 6 OD
values.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of
viability relative to the negative control, which is set at 100 %.
- classification according to UN GHS: if the mean percent tissue viability after 3 min-exposure is less than
(<) 50 % the test item is classified as corrosive (Category 1A); if the mean percent tissue viability after 3-
min-exposure is greater than or equal (≥) to 50 % AND < 15% after 60-min exposure the test item is class
ified as corrosive (Category 1B/1C); if the mean percent tissue viability after 60-min-exposure is ≥ 50 % af
ter 3-min exposure AND ≥ 15% after 60-min exposure the test item is considered as non-corrosive

The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro
reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability
after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100% concentration,
i.e. 50 μl per insert for 3 min. (room temperature) and 60 min. Cell viability was measured by the amount
of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)


NEGATIVE CONTROL
- Amount(s) applied: 50 µl
- Concentration (if solution): 0.9% NaCl in water

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 3 min

Value:

ca. 112

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not specified

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

exposure period: 60 min.

Value:

ca. 97

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not specified

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes, within regular intervals in the lab
- Acceptance criteria met for variability between replicate measurements: yes

Table 1: Tabular Summary of the results

Sample No.	Test item	Exposure time [min]	OD mean*	StdDev	% Viability
1 -3	Control NaCl 0.9%	60	1.92	0.18	100.00
4 -6	Norethisteron, gesiebt (D)	60	2.14	0.05	111.49
7 -9	Control NaCl 0.9%	3	2.22	0.07	100.00
10 -12	Norethisteron, gesiebt (D)	3	2.15	0.05	96.66

 *6 values

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 431 and EU Test Method B.40 bis. The mean value of cell viability was recorded to be 112 % (3 min. exposure) and 97 % (60 min. exposure). The test item was thus shown to be not corrosive to reconstructed human skin in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Analytical determination of stability and homogeneity of the test item in the vehicle was not performed specifically for this study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The usage of physiologic saline solution for formulation of the test item is plausible based on the current knowledge of the sponsor. No objections were seen particularly in regard to stability.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended by sonication ( 5 minutes) shortly prior to application in physiologic saline solution.
- Final preparation of a solid: 20% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): The preparation was visually described as suspension.

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 0.9% NaCl (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

no further incubation required

Number of animals or in vitro replicates:

3 cornea

Details on study design:

Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently (closed chamber method). The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers and the holes were sealed with tape.

Positive control: 20 % Imidazole in physiologic saline (w/v; 750 µL)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability:The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader (Bio-Tek EL 808, Software Gen5).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:decision criteria as indicated in the OECD TG 437

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-1.97

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Individual values of opacity, permeability and IVIS

Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group
				mean		SD
Vehicle control                    1	0.20	0.009	0.33
0.9 % NaCl                              2	-0.66	0.008	-0.53	-0.25		0.05
3	-0.61	0.005	-0.54
Positive Control                     4	49.46	1.305	69.04
20 % Imidazole                      5	63.93	1.317	83.68	80.93		10.78
6	71.10	1.265	90.07
Test item                                10	0.10	-0.001	0.09
20% Norethisteron,           11	-2.41	-0.001	-2.42	-1.97		1.87
gesiebt (D)                                  12	-3.56	-0.001	-3.57

 

Interpretation of results:

other: negative

Executive summary:

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be -1.97 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

- Principle of test:
Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (similar to EpiOcular).
The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (e.g. Alepee et. al., Toxicology in Vitro 34 (2016) 55–70).

GLP compliance:

yes (incl. QA statement)

Species:

other: human corneal epithelial cells

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthicTM Human Corneal Epithelial Model (HCE), SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per insert, plus 30 µl PBS to moisten and ensure good contact to the tissue

Duration of treatment / exposure:

60 min at room temperature

Duration of post- treatment incubation (in vitro):

16 hours in the incubator (37°C, 5% CO2, maximum humidity)

Number of animals or in vitro replicates:

three

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item. The test item is applied pure. Since the test item is a solution (ca. 40% in ethyl acetate) the solvent is tested in parallel to distinguish potential toxic effects of the test item from the effects of the solvent. After the post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction is performed. Cell viability is measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Irritation parameter:

other: cell viability (%)

Value:

ca. 103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Tabular Summary of the results

 

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	Negative control PBS	1.08	0.02	100.00
4 -6	Positive control SDS 0.3%	0.14	0.04	12.90
10 -12	Norethisteron, gesiebt (D)	1.11	0.06	103.15

*6 values

Interpretation of results:

other: negative

Executive summary:

The reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted with the test item. The undiluted test item was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured to be about 103% by the MTT conversion assay. Thus the test item is predicted as non-irritant under the conditions of this test method.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: Appendix B3 of "ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products", NIH Publication No. 10-7553, September 2010.

Principles of method if other than guideline:

- Principle of test: The Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) is a test method which implies the use of a complete tissue constituted of blood vessels and proteins that is capable of responding to chemical injury with an inflammatory process similar to the one occuring in the conjunctival tissue of the eye.

- Short description of test conditions: The test substance is applied directly to the chorioallantoic membrane (CAM) of fertilized chicken eggs

- Parameters analysed / observed: acute effects on haemorrhage, lysis of blood vessels and coagulation

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden
- Number of eggs: 4
- Characteristics of donor animals fertile Lohmann Brown hens
- Treatment conditions of eggs prior initiating testing: day 1-7: an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63%. day 8: with the large end
upward and not rotated for ensuring accessibility to the Chorioallantoic membrane (CAM) region
- Time interval prior to initiating testing: 8 days
- indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 μl/egg which corresponded to an amount from an average of 104 mg

Duration of treatment / exposure:

300 sec

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

4 eggs

Details on study design:

At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing. Undilutet test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL undiluted test item. 4 eggs each were used for the test item, negative and positive controls.

Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Scoring criteria for the acute effects and calculation of Irritancy Score (IS):

0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/ 300

H= observed start in seconds of haemorrhage reactions; L= observed start in seconds of vessel lysis, strong haemorrhage; C= observed start in seconds of blood - coagulation, albumen - coagulation

Data Interpretation:

Irritation Score (IS)

0-0.9 -> Non irritant
1-4.9 -> Slight irritant
5-8.9 -> Moderate irritant
9-21 -> Strong irritant

A test substance is considered to cause severe irritation when the IC value is greater than nine.

Irritation parameter:

other: irritation score (IS)

Run / experiment:

mean

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A test substance is considered to cause severe irritation when the IC value is greater than nine.

Table 1: Summary of results, test item

Egg	Effect	Effect detected after [sec]	Irritation Score (IS)
13	1	> 300
	2	> 300
	3	> 300	0
14	1	> 300
	2	> 300
	3	> 300	0
15	1	> 300
	2	> 300
	3	> 300	0
16	1	> 300
	2	> 300
	3	> 300	0

effect: 1 = vasodilatation, slight haemorrhage 2 = vessel lysis, strong haemorrhage 3 = blood-coagulation, albumen-coagulation

Interpretation of results:

other: negative

Executive summary:

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM. for 5 minutes. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

 

A study was performed for the assessment of the skin corrosion of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 431 and EU Test Method B.40 bis. The mean value of cell viability was recorded to be 112 % (3 min. exposure) and 97 % (60 min. exposure). The test item was thus shown to be not corrosive to reconstructed human skin in vitro.

 

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 110 %. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

 

Eye irritation

 

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be -1.97 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test system.

 

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM. for 5 minutes. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non irritant. The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

 

The reconstructed human corneal epithelial tissue-based in vitro test method (SkinEthic™ HCE) was conducted with the test item. The undiluted test item was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured to be about 103% by the MTT conversion assay. Thus the test item is predicted as non-irritant under the conditions of this test method.

Justification for classification or non-classification

Due to the results of the described studies no classification is required according to Regulation (EC) 1272/2008/EC (CLP), Annex I.