Reaction mass of sodium amino-bis{[4-(ethenylsubstituted)phenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-{[4-(ethenylsubstituted)phenyl]diazenyl}-{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate and polysodium amino-bis{[4-(ethenylsubstituted)-2-sulfonatophenyl]diazenyl}-hydroxynaphthalenesulfonate

Endpoint summary

Description of key information

The test substance was found to be not irritating as well as non corrosive to the skin and non irritating to eyes in respective in vitro tests.

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:

April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

None

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstituted human epidermis

Source strain:

not specified

Vehicle:

other: Dulbecco’s phosphate buffered saline (DPBS)

Details on test system:

EPISKIN Kit

Duration of treatment / exposure:

15 minutes

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

68.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: Assessment of Irritation Potential was based on the % Formazan production. Assessment was as follows: ≤ 50% of negative control- Irritant. > 50% of negative control - Non-irritant. Reversibility: other:. Remarks: The % viability of the test item was 68.1% and it was greater than 50% of the negative control. Hence under the conditions of the study the test item was found to be non-irritant to skin.

Other effects:

The negative controls mean OD was found to be 1.377, which is well within the acceptability range. The % viability of the positive control was 17.8% and it was lesser than 50% of the negative control, which reflects the irritant nature of positive control and the sensitivity of the tissues used in the study.

RESULTS

BLANK CORRECTED OPTICAL DENSITY (OD) VALUES FOR EACH TISSUE

Blank Corrected ODs
Replicate	NC	PC	Test item
Rep 1	NCR1a	PCR1a	4819R1a
	1.351	0.294	0.985
	NCR1b	PCR1b	4819R1b
	1.290	0.226	0.842
Rep 2	NCR2a	PCR2a	4819R2a
	1.505	0.232	0.946
	NCR2b	PCR2b	4819R2b
	1.439	0.257	1.112
Rep 3	NCR3a	PCR3a	4819R3a
	1.336	0.242	0.848
	NCR3b	PCR3b	4819R3b
	1.341	0.226	0.898

                       Mean OD of blank: 0.070

MEAN OPTICAL DENSITY (OD) VALUES FOR EACH TISSUE 

Mean ODs
Replicate	NC	PC	Test item
Rep 1	1.320	0.260	0.913
Rep 2	1.472	0.244	1.029
Rep 3	1.338	0.234	0.873
Mean	1.377	0.246	0.938
SD	0.083	0.013	0.081
%RSD	6.01	5.32	8.63

 

Key: Rep/R: Replicate; NC: Negative control; PC: Positive control; SD: Standard deviation; %RSD: Percent relative standard deviation; 4819= study number for test item FAT 40865/A TE

PERCENT VIABILITY (%)

% Viability
Replicate	PC	Test item
Rep 1	18.852	66.328
Rep 2	17.726	74.718
Rep 3	16.963	63.385
Mean	17.847	68.144
SD	0.950	5.881
% RSD	5.32	8.63

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be a non-irritant to skin.

Executive summary:

An in vitro study was performed in accordance to OECD Guideline 439 in compilance with GLP to assess the skin irritation potential of test item FAT 40865/A . The percent viability of the test item was 68.1% and it was greater than 50% of the negative control. Hence under the conditions of the study the test item was found to be a non-irritant to skin in accordance with UN GHS.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

None

Test system:

human skin model

Remarks:

EPISKIN

Source species:

human

Cell type:

other: Reconstituted Human Epidermis

Justification for test system used:

Recommended by guideline

Duration of treatment / exposure:

3 Minutes / 60 Minutes / 240 Minutes

Amount / concentration applied:

None

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

> 74.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3 Minutes application period result: The % viability was 86.4% ; 60 minutes application period :The % viability was 84.2%; 240 minutes application period: The % viability was 74.2%;

RESULTS

BLANK CORRECTED OPTICAL DENSITY (OD) VALUES FOR EACH TISSUE

	Blank Corrected ODs
	3 minutes application		60 minutes application		240 minutes application
Replicate	NC	Test item	NC	Test item	NC	PC	Test item
Rep 1	NCR1a	4818R1a	NCR1a	4818R1a	NCR1a	PCR1a	4818R1a
	0.942	0.745	1.085	0.938	0.854	0.152	0.835
	NCR1b	4818R1b	NCR1b	4818R1b	NCR1b	PCR1b	4818R1b
	0.828	0.668	0.849	0.758	0.739	-0.018	0.614
Rep 2	NCR2a	4818R2a	NCR2a	4818R2a	NCR2a	PCR2a	4818R2a
	0.823	0.762	0.666	0.531	0.786	0.026	0.521
	NCR2b	4818R2b	NCR2b	4818R2b	NCR2b	PCR2b	4818R2b
	0.858	0.806	0.772	0.613	0.886	0.022	0.589
Mean OD of blank	0.120		0.285		0.142

                   

Key: Rep/R: Replicate; NC: Negative control; PC: Positive control; SD: Standard deviation; 4818= study number for test item FAT 40865/A TE

 

MEAN OPTICAL DENSITY (OD) VALUES FOR EACH TISSUE

 

	Mean ODs
	3 minutes application		60 minutes application		240 minutes application
Replicate	NC	Test item	NC	Test item	NC	PC	Test item
Rep 1	0.885	0.707	0.967	0.848	0.797	0.067	0.725
Rep 2	0.841	0.784	0.719	0.572	0.836	0.024	0.555
Mean	0.863	0.746	0.843	0.710	0.817	0.046	0.640
SD	0.031	0.055	0.175	0.195	0.028	0.030	0.120
%RSD	3.65	7.35	20.80	27.48	3.42	66.46	18.73

 

Key: Rep/R: Replicate; NC: Negative control; PC: Positive control; SD: Standard deviation; %RSD: Percent relative standard deviation; 4818= study number for test item FAT 40865/A TE

PERCENT VIABILITY (%)

	% Viability
	3 minutes application	60 minutes application	240 minutes application
Replicate	Test item	Test item	PC	Test item
Rep 1	81.895	100.593	8.236	83.980
Rep 2	90.875	67.862	2.970	64.340
Mean	86.385	84.228	5.603	74.160
SD	6.350	23.144	3.724	13.888
%RSD	7.35	27.48	66.46	18.73

 

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

Conclusions:

Based on the results, the % viability result of the test item FAT 40865/A TE was 74.2% and it was greater than 35% of the negative control. Hence under the conditions of the study the test item was found to be ‘Non-corrosive’ to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals

Executive summary:

An OECD 431 guideline study was performed in accordance to GLP to assess the corrosion potential of test item FAT 40865/A . Test item, FAT 40865/A TE was applied topically to the EPISKIN^TM epidermal model (two epidermis units were used per each test item and negative controls) for three exposure periods viz., 3 minutes, 60 minutes and 240 minutes, additionally two epidermis units were used for positive control for the exposure period of 240 minutes. Exposure to the test item was terminated by rinsing with Dulbecco’s phosphate buffered saline (DPBS). The viability was assessed by incubating the tissues for 3 hours with MTT solution in a 12 well plate (0.3 mg/ml in assay medium; 2 ml per well). The precipitated formazan was then extracted using acidified isopropanol (0.5 ml) for 4 hours at room temperature and quantified spectrophotometrically at 560 nm using 96 well plates (200 μl/well). Glacial acetic acid and Sodium chloride (9.0 g/ L) treated epidermis were used as positive (PC) and negative controls (NC) respectively. For each treated tissue, the viability was expressed as the % of the negative control tissues (mean). 

3 minutes application:

The negative controls mean OD was found to be 0.863, which is well within the acceptability range (0.6 to 1.5). The variation within the replicates of negative control and test item was found to be lesser than 30%. The % viability of the test item was 86.4% and it was greater than 35% of the negative control. Hence no corrosive reaction observed with test item at 3 minutes application period.

 

60 minutes application:

The negative controls mean OD was found to be 0.843, which is well within the acceptability range (0.6 to 1.5). The variation within the replicates of negative control and test item was found to be lesser than 30%. The % viability of the test item was 84.2% and it was greater than 35% of the negative control. Hence no corrosive reaction observed with test item at 60 minutes application period

 

240 minutes application:

The negative controls mean OD was found to be 0.817, which is well within the acceptability range (0.6 to 1.5). The variation within the replicates of negative control and test item was found to be lesser than 30%. The % viability of the positive control was 5.6% and it was lesser than 50% of the negative control, which reflects the corrosive nature of positive control and the sensitivity of the tissues used in the study. The % viability of the test item was 74.2% and it was greater than 35% of the negative control. Hence no corrosive reaction observed with test item at 240 minutes application period.

 

The % viability of test item at the application periods of 3 minutes and 60 minutes showed greater than 35% mean tissue viability in terms of % negative control. Hence the test item is not categorized as corrosive 1A, 1B and 1C.

 

The % viability of test item at the application period of 240 minutes showed greater than 35% mean tissue viability in terms of % negative control. Hence the test item is categorized as ‘Non-corrosive’.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

The measurement of the opacity was performed with a photometer (570 nm) instead of an opacitometer. This can be seen as uncritical, because the opacity can be calculated from the absorption. The deviation was signed and assessed by the study director on 07. Aug. 2014.

GLP compliance:

yes

Specific details on test material used for the study:

None

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes (Bos primigenius Taurus (Fresh bovine corneas)) were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2 -4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

750 μL of the test item were tested as suspension at 20% concentration in 0.9% sodium chloride solution.

Duration of treatment / exposure:

Incubation time: 4 hours

Observation period (in vivo):

No data

Number of animals or in vitro replicates:

No data

Details on study design:

Refer to "Any other information on materials and methods incl. tables".

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

-0.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

None

Other effects:

None

 Opacity and Permeability Values

The absorption (570 nm) and opacity values which were measured before and after exposition are given in the following table:

Parameter	Negative Control
Absorption before exposition	0.1703	0.1803	0.1790
Absorption after exposition	0.2574	0.2851	0.5227
Opacity before exposition	1.4801	1.5146	1.5101
Opacity after exposition	1.8088	1.9280	3.3320
Opacity Difference	0.3287	0.4134	1.8219

 

Mean opacity difference of the negative control is 0.8547.

 

Absorption and Opacity Values Test Item and Positive Control

Parameter	Test ItemFAT 40865/A TE			Positive Control
Absorption before exposition	0.1908	0.2072	0.1664	0.1583	0.1376	0.1975
Absorption after exposition	0.2762	0.3804	0.4183	1.7073	1.6212	1.6112
Opacity before exposition	1.5517	1.6114	1.4669	1.4398	1.3728	1.5758
Opacity after exposition	1.8889	2.4010	2.6200	50.9683	41.8023	40.8507
Opacity Difference	0.3372	0.7897	1.1531	49.5285	40.4295	39.2750

 

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

Repl.	Negative Control			Test ItemFAT 40865/A TE			Positive Control
Meas.	0.0058	0.0065	0.0051	0.0022	0.0029	0.0045	0.3828	0.3462	0.4711
Corr.	0.0290	0.0325	0.0255	0.0110	0.0145	0.0225	1.9140	1.7310	2.3555
Mean	0.0290			--

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control 0.9% NaCl	0.764	1.290	61.6 %
	0.901
	2.204
Test Item FAT 40865/A TE	- 0.788	- 0.290	170.4 %
	- 0.283
	0.201
Positive Control 20% imidazole solution	76.949	71.790	8.5 %
	65.105
	73.318

 

Classification

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation

or serious eye damage.

IVIS	UN GHS Category Eye Damage
≤ 3	No Category
> 3;≤ 55	No prediction can be made
> 55	Eye damage Category I

 

In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category 1. The test item FAT 40865/A TE showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is - 0.290

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative or solvent control has to show an IVIS between 0 and 3.

The validity criteria and findings are given in the following table:

Parameter	Criterion	Found	Assessment
IVIS of negative control 0.9% NaCl	0 - 3	1.290	ok
IVIS of positive control 20% imidazole solution	35.6 – 130.4	71.790	ok

 Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Interpretation of results:

not irritating

Conclusions:

Under the conditions of this test, the test item FAT 40865/A TE showed no effects on the cornea of the bovine eye. Hence, it was considered to be non-irritating to eyes.

Executive summary:

An OECD 437 guideline study was performed in accordance to GLP to assess the irritation potential of test item FAT 40865/A to eyes using bovine corneas. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item FAT 40865/A TE was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea. 20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea. The calculated IVIS (in vitro irritancy score) was - 0.290. According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS≤3 requires no classification for eye irritation or serious eye damage. Hence, under the conditions of this test system, the test item FAT 40865/A TE showed no effects on the cornea of the bovine eye and considered to be non-irritating to eyes.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three key in vitro tests were performed as per OECD test guideline to evaluate irritation and corrosion potential of FAT 40865/A TE.

An OECD 423 guideline study was performed in accordance to GLP to assess the skin corrosion potential of test item FAT 40865/A using EPISKIN epidermal model for three exposure periods viz., 3 minutes, 60 minutes and 240 minutes. Based on the results, the % viability result of the test item FAT 40865/A TE was 74.2% and it was greater than 35% of the negative control. Hence under the conditions of the study the test item was found to be ‘Non-corrosive’ to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemicals.

An OECD 439 guideline study was performed in accordance to GLP to assess the skin irritation potential of test item FAT 40865/A . The percent viability of the test item was 68.1% and it was greater than 50% of the negative control. Hence under the conditions of the study the test item was found to be Non-irritant to skin in accordance with UN GHS as specified in the OECD Guideline for the Testing of Chemical.

An OECD 437 guideline study was performed in accordance to GLP to assess the irritation potential of test item FAT 40865/A to eyes using bovine corneas. They were collected from slaughtered cattle which were between 12 and 60 months old. The test itemFAT 40865/A TEwas brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the cornea. 20% Imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea. The calculated IVIS (in vitro irritancy score) was - 0.290. According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤3 requires no classification for eye irritation or serious eye damage. Hence, under the conditions of this test system, the test item FAT 40865/A TE showed no effects on the cornea of the bovine eye and considered to be non-irritating to eyes.

 

Justification for classification or non-classification

The test substance was not irritating as well as non corrosive to skin and non irritating to eyes. Hence, it does not require classification for irritation/corrosion as per the CLP (Regulation 1272/2008) criteria.