Registration Dossier

EYE IRRITATION
Hopf 1978: Primary skin irritation test, German Society for Fat Science and the Cosmetics Commission of the Federal Health Office Guidelines for testing of personal care products, Not irritating - 50 % test material in olive oil (rabbits)
Dreher 2014: in vitro skin irritation test (BCOP), OECD 437 and EU Method B.47 (GLP), Negative
SKIN IRRITATION
Hopf 1978: Acute eye irritation test, German Society for Fat Science and the Cosmetics Commission of the Federal Health Office Guidelines for testing of personal care products, Not irritating - 50 % test material in olive oil applied daily for 5 days (male and female guinea pigs)
Dreher 2014: in vitro skin irritation test (EpiSkin), OECD 439 and EU Method B.46 (GLP), Negative

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 June 2014 to 9 Decembre 2014

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)

Deviations:

yes

Remarks:

43 hour incubation period was used instead of 42 hours specified in the protocol.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

43 hour incubation period was used instead of 42 hours as specified in the protocol.

GLP compliance:

yes

Specific details on test material used for the study:

Batch : SC00010875
Expiration Date: 09 April 2015

Test system:

human skin model

Source species:

human

Cell type:

other: EpiSkinTM three-dimensional human skin model

Cell source:

other: A reconstructed epidermis with a functional stratum corneum, supplied by SkinEthic Laboratories, Lyon, France.

Vehicle:

unchanged (no vehicle)

Remarks:

The test article was used as supplied.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

A volume of 40 μL of the undiluted test article was added topically to the tissues. A volume of 40 μL of the positive and negative control solutions was used.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.The tissues were then incubated at 37°C for 43 hours

Number of replicates:

The test was performed on a total of three tissues per test article, negative and positive control.

Irritation / corrosion parameter:

% tissue viability

Value:

57.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The group mean viability for the negative control was 100.0 %
The group mean viability for the positive controls was 11.8 %

Table 1: Results

Substance	Tissue replicate	OD570		Corrected mean	Standard deviation	Coefficient of variance	% Relative survival
Substance	Tissue replicate	Aliquot 1	Aliquot 2	Corrected mean	Standard deviation	Coefficient of variance	Tissue	Mean
Negative control	A	1.022	1.041	1.031	9.6940	9.694	93.3	100.0
	B	1.068	1.046	1.057			95.6
	C	14.221	1.236	1.228			111.1
Test material	A	0.805	0.775	0.790	11.9747	20.766	7.5	57.7
	B	0.556	0.563	0.560			50.6
	C	0.562	0.563	0.563			50.9
Positive control	A	0.136	0.141	0.139	2.0100	16.984	12.5	11.8
	B	0.106	0.105	0.106			9.6
	C	0.152	0.145	0.148			13.4
Blank		0.001	-0.001

Interpretation of results:

GHS criteria not met

Remarks:

Not sufficient to be classified under the GHS criteria

Conclusions:

The test article, Adoxal, was considered to be non-irritant in the in vitro skin model EpiSkin.

Executive summary:

In the in vitro study (Dreher, 2014), the potential for the test material to cause skin irritation was investigated in a study conducted in accordance with standardised guideline OECD 439 and EU Method B.46 under GLP conditions using the EpiSkin in vitro model.

EpiSkinTM inserts were treated with the test material, negative control and positive control for 15 minutes. At the end of the exposure period, the tissues were washed and assessed for viability using MTT (3 -(4,5 -Dimethylthiazol-2 -yl)-2,5 -diphenyl tetrazolium bromide).

The group mean viability for the test material was 57.7 %. The group mean viability for the negative control was 100.0 % and 11.8 % for the positive control.

The test material was determined to be non-irritant under the conditions of the test in the in vitro skin model EpiSkin.

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2014 to 15 December 2014

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch SC00010875
Expiration date 09 April 2015

Species:

other: Bovine eyes

Strain:

other: Corneas

Details on test animals or tissues and environmental conditions:

The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL) in a suitably sized container and transported on the same day to the testing facility.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: Undiluted

Duration of treatment / exposure:

10 minute incubation at 32°C ± 1°C.

Duration of post- treatment incubation (in vitro):

After the 1st incubation (10 minute incubation at 32°C ± 1°C) with exposure (750 µL) , each cornea was washed with media containing phenol red followed by media without phenol red and the corneas assessed for opacity. The corneas were then incubated (horizontally) for 2 hours ± 10 minutes after which, corneal opacity was measured.

Number of animals or in vitro replicates:

Three corneas

Irritation parameter:

in vitro irritation score

Value:

1.03

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corneal Opacity :
The mean corrected opacity reading for the test article was 1.0.
The mean corrected opacity reading for the negative control was 0.0.
The mean corrected opacity reading for the positive control was 35.3.

Corneal Permeability:
The mean group corrected optical density for the test article was 0.002.
The mean group corrected optical density for the negative control was 0.000.
The mean group corrected optical density for the positive control was 0.183

The assay was considered valid as the assay acceptance criteria were met.

Table 1: Corneal Opacity

Test substance	Cornea No.	Initial Opacity	Post Incubation Opacity	Change in Opacity	Mean Change in Opacity	Corrected Opacity	Mean Corrected Opacity
Test material	2	3	4	1	N/A	-0.3	1.0
	5	3	7	4		2.7
	15	3	5	2		0.7
NaCl (negative control)	6	2	3	1	1.33	-0.3	0.0
	17	2	4	2		0.7
	21	2	3	1		-0.3
DMF (positive control)	16	3	52	49	N/A	47.7	35.3
	18	3	22	19		17.7
	24	3	45	42		40.7

Table 2: Corneal Permeability

Test substance	Cornea No.	Mean Blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490
Test material	2		0.022	0.022	N/A	0.003	0.002
	5		0.021	0.021		0.002
	15		0.019	0.019		0.000
NaCl (negative control)	6	0.000	0.021	0.021	0.019	0.002	0.000
	17		0.016	0.016		-0.003
	21		0.019	0.019		0.000
DMF (positive control)	16		0.163	0.163	N/A	0.144	0.183
	18		0.168	0.168		0.149
	24		0.273	0.273		0.254

Table 3: IVIS scores

Test substance	IVIS
Test material	1.03
NaCl (negative control)	0.00
DMF (positive control)	38.07

Interpretation of results:

not classified

Conclusions:

The test article, Adoxal, produced an In Vitro Irritancy Score (IVIS) of 1.03 and does not require classification for eye irritation.

Executive summary:

In the in vitro study (Dreher, 2014), the potential of the test material to cause serious eye irritation was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using bovine corneas.

750 µL of undiluted test material was applied to each of three corneas and incubated for 10 minutes at 32 °C ± 1 °C. After incubation, each cornea was washed with media containing phenol red followed by media without phenol red and incubated horizontally for 2 hours ± 10 minutes and then measured for opacity.

Permeability of the corneas was measured by replacing the media in the anterior chamber with 1 mL of 4 mg/mL sodium fluorescein. The posterior chamber was filled with fresh media. The corneas were incubated vertically for 95 minutes at 32 °C ± 1 °C. The posterior chambers were the removed and three 350 µL aliquots of the media were taken for analysis of optical density at 490 nm.

The mean corrected opacity reading for the test material was determined to be 1.0. The mean corrected opacity reading for negative and positive controls was 0.0 and 35.3, respectively. The corneas treated with the positive control were cloudy following treatment.

The mean corrected optical density for the test material was determined to be 0.002. The mean corrected optical densities for negative and positive controls were 0.000 and 0.183, respectively.

Under the conditions of the test, the test material was determined to not require classification for eye irritation. The test material produced an in vitro irritancy score (IVIS) of 1.03 in bovine corneas.

Endpoint conclusion:

no adverse effect observed (not irritating)

Endpoint conclusion:

no study available

SKIN IRRITATION/CORROSION

As the available in vitro study is suitable for the purposes of classification and labelling and qualitative risk assessment, further animal studies are not necessary.

In the in vivo study (Hopf, 1978), the irritation potential of the test material was determined in a primary skin irritation test performed according to the recommendations of the German Society for Fat Science and the Cosmetics Commission of the Federal Health Office Guidelines for testing of personal care products. The study pre-dates GLP.

Twelve Pirbright white guinea pigs, 6 males and 6 females, weighing 300 - 500 g were topically treated on the shorn flank daily with 50 % test material in olive oil for 5 days. The test solution was rubbed in for 30 seconds at each application. Animals were individually housed and provided feed and water ad libitum. The room temperature was 20 °C with a humidity of 50 % and a dark/light cycle of 12 hours.

Under the conditions of the test no dermal irritation was recorded in any of the treated animals. The test material was determined to be not irritating.

In the in vitro study (Dreher, 2014), the potential for the test material to cause skin irritation was investigated in a study conducted in accordance with standardised guideline OECD 439 and EU Method B.46 under GLP conditions using the EpiSkin in vitro model.

EpiSkinTM inserts were treated with the test material, negative control and positive control for 15 minutes. At the end of the exposure period, the tissues were washed and assessed for viability using MTT (3 -(4,5 -Dimethylthiazol-2 -yl)-2,5 -diphenyl tetrazolium bromide).

The group mean viability for the test material was 57.7 %. The group mean viability for the negative control was 100.0 % and 11.8 % for the positive control.

The test material was determined to be non-irritant under the conditions of the test in the in vitro skin model EpiSkin.

EYE IRRITATION

As the available in vitro study is suitable for the purposes of classification and labelling and qualitative risk assessment, further animal studies are not necessary.

In the in vivo study (Hopf, 1978), the irritation potential of the test material was determined in an acute eye irritation study performed using rabbits according to the Draize method. The study was performed according to the recommendations of the German Society for Fat Science and the Cosmetics Commission of the Federal Health Office Guidelines for testing of personal care products. The study pre-dates GLP.

A 50 % solution of the test material in olive oil was instilled into the conjunctival sac of one eye in each of the 8 test rabbits, and the lid was held closed for 1 minute. The other eye was treated with water and served as a control. Washing was performed after 1 minute in half of the treated animals.

Under the conditions of the test no eye irritation was recorded in any of the treated rabbits. The test material was determined to be not irritating.

In the in vitro study (Dreher, 2014), the potential of the test material to cause serious eye irritation was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions using bovine corneas.

750 µL of undiluted test material was applied to each of three corneas and incubated for 10 minutes at 32 °C ± 1 °C. After incubation, each cornea was washed with media containing phenol red followed by media without phenol red and incubated horizontally for 2 hours ± 10 minutes and then measured for opacity.

Permeability of the corneas was measured by replacing the media in the anterior chamber with 1 mL of 4 mg/mL sodium fluorescein. The posterior chamber was filled with fresh media. The corneas were incubated vertically for 95 minutes at 32 °C ± 1 °C. The posterior chambers were the removed and three 350 µL aliquots of the media were taken for analysis of optical density at 490 nm.

The mean corrected opacity reading for the test material was determined to be 1.0. The mean corrected opacity reading for negative and positive controls was 0.0 and 35.3, respectively. The corneas treated with the positive control were cloudy following treatment.

The mean corrected optical density for the test material was determined to be 0.002. The mean corrected optical densities for negative and positive controls were 0.000 and 0.183, respectively.

Under the conditions of the test, the test material was determined to not require classification for eye irritation. The test material produced an in vitro irritancy score (IVIS) of 1.03 in bovine corneas.

Justification for selection of skin irritation / corrosion endpoint:
The in vitro study was selected on the basis that it was performed in accordance with standardised guidelines, with complete reporting and it is sufficient for the purpose of classification and labelling.

Justification for selection of eye irritation endpoint:
The in vitro study was selected on the basis that it was performed in accordance with standardised guidelines, with complete reporting and it is sufficient for the purpose of classification and labelling.

Skin

In accordance with with criteria for classification and labelling as defined in Annex I of Regulation (EC) No 1272/2008 (CLP), the test material does not require classification for skin irritation and corrosion.

Eye

In accordance with the criteria for classification as defined in Annex I of Regulation 1272/2008 (CLP), the test material does not require classification for eye irritation.