Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Endpoint summary

Description of key information

An in vitro-study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 90.7 % after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

The test item was applied by instillation of 0.1 mL into the right eye of each of three young adult New Zealand White rabbits. No abnormal findings were observed in the cornea of any animal at any of the examinations. No clinical signs of toxicity were observed. The test item did not induce significant or irreversible damage to the rabbit eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2016 until 18 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 439 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS (2003, last rev. 2015)

Deviations:

no

Principles of method if other than guideline:

Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS prior to application, and spread to match the surface of the tissue

Duration of treatment / exposure:

60 minutes

Species:

other: reconstituted human epidermis model

Type of coverage:

other: Topical

Preparation of test site:

other: Not applicable

Vehicle:

other: No vehicle used

Amount / concentration applied:

Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 µL DPBS prior to application, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.

Duration of treatment / exposure:

60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Irritation / corrosion parameter:

% tissue viability

Remarks:

Episkin model, 3 tissues per concentration

Run / experiment:

mean

Value:

90.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results after treatment with the test item and the controls after 60 minutes exposure interval

Dose Group	Tissue No.	well 1	well 2	well 3	Mean	Mean-Blank*	Mean of three tissues	Stand. Dev.	Rel. Absor-bance [%] Tissue 1, 2 + 3**	Stand. Dev. [%]	Mean Rel. Ab-sorbance [% of Negative Control] ***	Dif-ference 1****	Dif-ference 2*****	Dif-ference 3 ******	Mean OD blank corrected viable tissues without MTT	Mean OD of two killed tissues blank cor-rected treated with the test item	Mean OD of two killed tissues blank corrected treated with negative control	Cor-rected Via-bility Mean Abs. 1 [%]	Corrected Via-bility Mean Abs. 2 [%]	Corrected Via-bility Mean Abs. 3 [%]
Blank		0.037	0.038	0.038	0.038	0.000
Nega-tive Control	1	1.892	1.879	1.888	1.886	1.848	2.019	0.181	91.5	9.0	100.0
	2	2.037	2.064	2.013	2.038	2.000			99.1
	3	2.244	2.243	2.252	2.246	2.209			109.4
Test Item	1	1.740	1.748	1.771	1.753	1.715	1.852	0.121	84.9	6.6	91.8	1.848	1.836	1.832	0.004	0.155	0.139	91.553	90.935	90.737
	2	1.867	1.971	1.966	1.935	1.897			94.0
	3	1.957	1.990	2.003	1.983	1.945			96.4
Posi-tive Control	1	0.104	0.112	0.107	0.108	0.070	0.074	0.004	3.5	5.4	3.7
	2	0.109	0.125	0.112	0.115	0.078			3.8
	3	0.113	0.114	0.113	0.113	0.076			3.8

 

*             Mean of three replicate wells after blank correction

**            relative absorbance per tissue [rounded values]:

***           relative absorbance per treatment group [rounded values]:

 

****        Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus mean OD (three replicates one viable tissue without MTT after blank correction)

*****       Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus (mean OD of two killed tissues treated with test item minus mean OD of two killed tissues treated with negative control)

******      Mean OD viable Tissue (blank corrected from three tissues with three replicates) minus mean OD (three replicates one viable tissue without MTT after blank correction) minus (mean OD of two killed tissues treated with test item minus mean OD of two killed tissues treated with negative control)

Interpretation of results:

other:

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

An additional test with one viable tissue had to be performed, due to the intensive black colour of the test item. Since the test item dyed the MTT-solution black in the pre-test for direct MTT reduction also due to its intensive black colour, a possible colour turn into blue or purple as a sign being a MTT reducer was not possible to observe. Therefore, as a precaution, an additional test with freeze-killed tissues was performed.

The test item, the negative control (DPBS) and the positive control (5% SLS) were applied to each of triplicate tissue.

The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following approximately 68.5 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.7% thus ensuring the validity of the test system.

The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were 9% at the highest (threshold of the "OECD Guideline for the Testing of Chemicals 439:In vitroSkin Irritation: Reconstructed Human Epidermis Test Method”: < 18%), thus ensuring the validity of the study.

Compared to the relative absorbance value of the negative control the corrected mean relative absorbance value was reduced to 90.7% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

in vivo study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 04 July 2016 and 22 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.35 or 3.23 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.
Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

0.1 mL of the test item, which was found to weigh approximately 94 mg

Duration of treatment / exposure:

Up to 1 hour

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 rabbits

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item, which was found to weigh approximately 94 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Annex 2.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
After consideration of the ocular responses produced in the second treated animal, a third animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Annex 3.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
An additional observation was made on Day 7 to assess the reversibility of the ocular effects.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

cornea opacity score

Basis:

animal: 75495 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal: 75504 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal: 75515 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 75495 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.3

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

animal: 75504 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 75515 Female

Time point:

other: Mean at 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

other: redness

Basis:

animal: 75495 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

other: redness

Basis:

animal: 75504 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

other: redness

Basis:

animal: 75515 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.7

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 75495 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 75504 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 75515 Female

Time point:

other: mean at 24, 48 and 72 hours

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Ocular Reactions
Individual and mean scores for ocular irritation are given in Appendix 1.
Blue colored staining of the fur around the treated eye, the inner eyelids and conjunctival membrane was noted in all animals at all observations.
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye at the 24-Hour observation. No iridial effects were noted in two treated eyes during the study.
Moderate conjunctival irritation was noted in all treated eyes 1 hour after treatment and at the 24 and 48 Hour observations. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in two treated eyes at the 72 Hour observation.
All treated eyes appeared normal at the 7 Day observation.
No corrosive effects were noted during the study. The test item did not induce significant or irreversible damage to the rabbit eye.

Other effects:

Body Weight
Individual body weights and body weight change are given in Appendix 2.
One animal showed body weight loss and the other two animals showed expected gain in body weight during the study.

Appendix 1     Eye Irritation Scores

Individual Scores for Eye Irritation

								Conjunctivae
Rabbit Number and Sex	IPR	Evaluation interval	Corneal Opacity	Area of Corneal Opacity	Iris	Redness	Chemosis		Discharge
75493Female	0	1 Hour	0	0	0	2	1		2Sf
75498Male	0		0	0	0	2	1		1Sf
75493Female		24 Hour	0	0	0	1	1		1Sf
75498Male			0	0	0	1	1		1Sf
75493Female		48 Hour	0	0	0	1	0		0Sf
75498Male			0	0	0	1	0		0Sf
75493Female		72 Hour	0	0	0	0	0		0Sf
75498Male			0	0	0	0	0		0Sf

IPR=Initial pain reaction

Sf =       Red/orange colored staining of the fur around the treated eye

Mean Values after 24, 48 and 72 Hours

				Conjunctivae
Rabbit Number and Sex	Number of available data points	Corneal Opacity	Iris	Redness	Chemosis
75493Female	3	0.0	0.0	0.7	0.3
75498Male	3	0.0	0.0	0.7	0.3

Assessment According to Regulation (EC) No. 1272/2008

			Conjunctivae
Evaluated Intervals	Corneal Opacity	Iris	Redness	Chemosis
24 Hours	Not classified	Not classified	Not classified	Not classified
48 Hours	Not classified	Not classified	Not classified	Not classified
72 Hours	Not classified	Not classified	Not classified	Not classified

Appendix 2     Individual Body Weights and Body Weight Change

	Individual Body Weight (kg)
Rabbit Number and Sex	Day 0	Day 3	Body Weight Change (kg)
75493Female	2.38	2.33	-0.05
75498Male	3.48	3.52	0.04

Interpretation of results:

other: does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Conclusions:

According to the findings in this study, the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Executive summary:

The primary eye irritation potential of the test item was investigated according to a method compatible with OECD test guideline No. 405 and Method B5. The test item was applied by instillation of 0.1 mL into the right eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48, 72 hours and 7 days after test item instillation. 

The mean score was calculated separately for each animal across three scoring times (24, 48 and 72 hours after instillation) for corneal opacity, iritis, redness and chemosis of the conjunctivae. The individual mean score for corneal opacity was 0.0 for all treated eyes. The individual mean scores for iritis were 0.3 for the first treated eye and 0.0 for the second and third treated eyes. The individual mean scores for conjunctival reddening for the first treated eye were 2.0 and 1.7 for the second and third treated eyes. The individual mean scores for conjunctival chemosis were 1.3 for all treated eyes. 

The instillation of the test item into the eye resulted in conjunctival irritation. These effects were reversible and were no longer evident 7 days after treatment in all animals (end of the observation period). No abnormal findings were observed in the cornea of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No clinical signs of toxicity were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

According to the findings in this study, the test item does notmeet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification.

Criteria for classification not met.