Alkali salt of sulfonated aryl azo amino sulfonyl aryl azo sulfonyl aryl azo aryl sulfonate

Endpoint summary

Description of key information

Reactive Brown DYHY 0331/0334 is not iriitating to skin or eyes

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:

26 June 2014 to 11 August 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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Details on test animals or test system and environmental conditions:

Test System: EPISKIN™ - 0.38 cm2
Supplier: SkinEthic Laboratories (4, A. Fleming – 69007 Lyon –France).
Batch: 14-EKIN-028

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2).

Duration of treatment / exposure:

exposure period of 15 minutes followed by a 42 ± 1 hour recovery period.

Number of animals:

Number of replicates: 3

Details on study design:

Positive control item: Sodium Dodecyl Sulphate (SDS) (SIGMA, batch 031M0035V), diluted at the final concentration of 5% (w/v) in sterile water
for injection (Baxter, batch 13A3106).

Negative control item: D-PBS (GIBCO, batch 1453554).

Non specific colour (NSC) control: test item treated tissues without MTT.

Non specific MTT reduction (NSMTT): killed tissues treated with the test item.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean cell viability

Run / experiment:

Time point: 42 h

Value:

116.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The Non Specific Colour (NSC), relative to the D-PBS Control, was 0.2%, lower than 5%. The non specific MTT reduction induced by the test item,
was 4.7% when compared to the negative control performed with alive tissues. Based on these results, no correction of the % viability obtained for the alive test item treated tissues was performed.
The test item did not induce cell death in any replicate with a mean cell viability of 116.6% when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 14 lower than 18).

Interpretation of results:

GHS criteria not met

Conclusions:

The test item should be considered not irritant.

Executive summary:

The potential of the test item Reactive Brown DYHY0331/0334 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 +/- 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a blue/purple coloured solution was observed at the end of the incubation period, indicating that the test item interacted directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a dark blue coloured solution was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added in the Main Assay. In Main Assay the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.3). Based on the stated criteria (mean viability <40% and SD of % viability <18), the assay was regarded as valid.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using one alive treated tissue without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using killed tissues and compared with negative control performed with alive tissues. Based on the results obtained (both NSC and NSMTT < 5%), no correction of the % viability obtained for the alive treated tissues was performed.

The test item did not induce cell death in any replicate with a mean cell viability of 116.6% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 14 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item Reactive Brown DYHY0331/0334 is classified as not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 September 2014 to 22 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’Arbresle Cedex, France
- Age at study initiation: approximately 10 months
- Weight at study initiation: approximately 5 kg
- Housing: 1 per cage (during the study)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 7 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C+/-2 °C
- Humidity (%): 55%+/-15%
- Air changes (per hr): Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: 30 September2014 To: 22 October 2014

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:

Only once. Exposure for 1 hour.

Observation period (in vivo):

1, 24, 48, 72 hours, days 7, 14, 21 after dosing.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 1 hour

SCORING SYSTEM: Irritation to the cornea, iris and conjunctivae were assigned a numerical value according to the Draize scale.

TOOL USED TO ASSESS SCORE: macroscopic observation.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

> 0 - < 1

Max. score:

3

Reversibility:

fully reversible within: 48 hours after dosing

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Slight redeness of conjuntivae (score of 1) observed at 1 and 24 hours after dosing in all treated animals. Slight chemosis of conjuntivae (score of 1) observed at 1 hour after dosing in all treated animals.

Other effects:

Dark staining of the conjunctivae was observed on Day 1 in one animal. This sign was no longer apparent at 48 hour post-dose observation.
Dark staining of the conjunctivae was also noted in the remaining two animals up to Day 14 post-dose (Day 15 of the study).

Clinical signs

A dark staining of the fur around the treated eye was noted in all animals up to the observation performed on Day 22 of the study. This sign was related to the dark colour of the substance.

There was no indication of a systemic effect.

Pain/distress signs

No signs of pain or distress were observed in the animals during the study.

Body weight

Changes in body weight, seen during the course of the study, were not remarkable.

Interpretation of results:

GHS criteria not met

Conclusions:

No remarkable signs of irritation were recorded in any treated animal during the observation period.

Executive summary:

The acute eye irritation of Reactive Brown DYHY 0331/0334 was investigated in rabbits.

A 0.1 g aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours, 7, 14 and 21 days after dosing.

Slight redeness of conjuntivae (score of 1) observed at 1 and 24 hours after dosing in all treated animals. Slight chemosis of conjuntivae (score of 1) observed at 1 hour after dosing in all treated animals.

There were no signs of pain/distress after dosing.

Changes in body weight were not remarkable.

There was no indication of a systemic effect related to treatment.

These results indicate that the test item has no remarkable effects on the eye of the rabbit.

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would suggest the following:

Classification: Not required

Signal word: None indicated

Hazard statement: None indicated

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of the test item Reactive Brown DYHY0331/0334 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 +/- 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor. Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se; a blue/purple coloured solution was observed at the end of the incubation period, indicating that the test item interacted directly with MTT. In a second step, the test item was assayed for the ability of colouring water per se; a dark blue coloured solution was observed, which indicates a colouring capacity of the test item. Based on these results, additional controls were added in the Main Assay. In Main Assay the test item was applied as supplied in three replicates at the treatment level of 20 mg/epidermis unit, each measuring 0.38 cm2 (treatment level: 53 mg/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 2.3). Based on the stated criteria (mean viability<40% and SD of % viability <18), the assay was regarded as valid.

In order to verify if the test item results had to be corrected, the non specific colour (NSC) was evaluated using one alive treated tissue without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using killed tissues and compared with negative control performed with alive tissues. Based on the results obtained (both NSC and NSMTT < 5%), no correction of the % viability obtained for the alive treated tissues was performed.

The test item did not induce cell death in any replicate with a mean cell viability of 116.6% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 14 (lower than 18, as stated in the Study Protocol). Based on the results obtained, the test item Reactive Brown DYHY0331/0334 is not irritating to the skin.

In the acute dermal toxicity study in rats at the limit dose of 2000 mg/kg body weight, no adverse systemic or local effects were noted.

The potential of the test item, Reactive Brown DYHY0331/0334, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on 26 July 2013), the Guidance Document OECD series on testing and assessment no. 160 and the method described in the document issued by U.S. EPA/OPP (31 May 2013). The test item was dissolved at 20% (w/v) in physiological saline and tested on the epithelial surface of three idoneous bovine corneas, for an exposure period of 4 hours. Positive and negative controls [a 20% (w/v) Imidazole solution in physiological saline and physiological saline alone, respectively] were concurrently tested in the same number of replicates. The mean opacity detected with an opacimeter at the end of the test item exposure period was 67.0. This was confirmed at the macroscopic observation in which opacity of the treated corneas was noted although the treated corneas appeared coloured; slight exfoliation was also observed in one of the treated corneas. After the determination of opacity, the epithelial surface was treated with a 0.5% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability. The calculated mean permeability OD490 value of the corneas treated with the test item was 6.045. The results obtained indicate that the test item induced changes in cornea opacity and permeability under the reported experimental conditions. The calculated in vitro irritancy score (IVIS) for the test item was 157.7. Negative and positive controls gave the expected results.

According to this test result the item Reactive Brown DYHY0331/0334 should be classified as corrosive or severely irritant to the eye, however, this is regarded as false-positive result due to formation of the more reactive alcohol in the testing solution.

In the long history of developing reactive dyes, none of them were a severe eye irritant other than by causing a long-lasting discoloration of the eye. In addition, the in-vitro test for skin irritation was clearly negative and no adverse local effects were observed during the acute dermal test in rats. Taking into account that the test substance was dissolved in water for the BCOP assay and hydrolyses rapidly to the vinyl and further to the alcohol form of the test substance and knowing that the BCOP assay has a high rate of false positive results with ketones and alcohols, this test was disregarded as invalid due to a false positive result.

As a consequence, it was decided to test Reactive Brown DYHY 0331/0334 in one rabbit eye first and closely monitored this rabbit for any adverse effects. As no irritating effects were noted in this animal two further rabbits were used to complete the in-vivo eye-irritation testing.

For this in-vivo eye irritation study in rabbits, a 0.1 g aliquot of the test item was introduced into the right eye of a total of 3 animals. The resulting reaction to treatment was assessed approximately 1, 24, 48, and 72 hours, 7, 14 and 21 days after dosing.

Slight redness of conjunctivae (score of 1) observed at 1 and 24 hours after dosing in all treated animals. Slight chemosis of conjunctivae (score of 1) observed at 1 hour after dosing in all treated animals. There were no signs of pain/distress after dosing.

Changes in body weight were not remarkable and there was no indication of a systemic effect related to treatment.

These results indicate that the test item has no irritating effects on the eye of the rabbit.

Reactive Brown DYHY 0331/0334 is therefore regarded as not irritating to skin and eyes.

Justification for classification or non-classification

No classification necessary