Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Description of key information

Two studies on skin irritation are available, one in vitro (key) study and one in vivo (supporting) study. Both studies conclude that Reactive Red 45:1 is not irritating to the skin.

Three studies on eye irritation are available, two in vitro studies and one in vivo study. Based on the first in vitro study (BCOP assay), no conclusion on classification could be drawn. A second in vitro study (EpiOcular assay) showed a positive effect but no conclusion on classification could be drawn either. Therefore, finally an in vivo study was performed in rabbits, which concluded that Reactive Red 45:1 is not eye irritant.

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:

19.04-29.07.2016

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:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: model EpiDermTM
- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23329)
- Date of initiation of testing: 19.04.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 g

NEGATIVE CONTROL
PBS (phosphate buffered saline) prepared 25/02/2016, exp. 25/08/2016 and prepared 23/03/2016 exp. 23/09/2016
- Amount(s) applied: 25 µL


POSITIVE CONTROL
5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 012616TMB, exp. 26/01/2017

Duration of treatment / exposure:

60 min.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

54.3

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test item Reactive Red 45:1 was 54.3 % of negative control average value. The measured value was then corrected, due to red colour of the test substance.
Corrected viability of treated tissues was 53.4 %, i.e. viability was > 50 %.

Executive summary:

The test item, Reactive Red 45:1, was assayed for the in vitro skin irritation
in human epidermal model EpiDerm^TM.The test was performed according to the OECD Test Guideline No.439: In VitroSkin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

After pre-incubation of tissues, 25 mg of the test item was placed directly a top to the previously moistened tissue and it was spread on the entire tissuesurface. Length of exposition was 60 minutes. Three tissues were used for the test item and every control.

After removal of the test substance, tissues were post-incubated for approximately
42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

At direct reduction assay in a test tube MTT medium was coloured red, so direct MTT reduction was excluded.

In the first experiment too high differenceamong viability of tissues was observed in range close to cut off value. The experiment was then repeated. In the repeated experiment all test conditions were fulfilled and average viability of treated tissues was54.3% (53. 4 % after correction),i.e. viability was >50 %.

The effect of the test item was negative in EpiDerm^TMmodel (tissues were not damaged).

According to the classification criteria given in chapter 4.5., the test item, Reactive Red 45:1, is considered to have no category in regard to skin irritation.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August-November 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards and acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

The study report contains the limited information on the test conditions because the study has been performed in 1976 . However available information is sufficient to conclude on the classification of the substance. Further the test was performed on the vertebrates and use of results from old experimental studies is one of the options to provide information requested by REACH. New experimental studies with vertebrates must only be conducted if there is no adequate existing information.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Data are not available

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

0.5 grams test material was wetted with water

Duration of treatment / exposure:

4h

Observation period:

24h

Irritation parameter:

overall irritation score

Basis:

animal:

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating.

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:

01.-02.06.2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

TEST MATERIAL: Bovine eyes
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech
Republic
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 μl of suspension
- The test substance was tested as suspension prepared from test substance at 20% concentration in a
0.9% sodium chloride solution.
2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

1.5h

Number of animals or in vitro replicates:

Exposed group (test substance) - 3 corneas (No. 9, 10, 12)
Positive control group (20% Imidazole) – 3 corneas (No. 13, 14, 15)
Negative control group (0.9% NaCl) – 3 corneas (No. 6, 8, 11)

Details on study design:

TEST PROCEDURE
- Selection of corneas, mounting in holders
- incubation with EMEM 1 hour (32 ± 1°C)
- removed EMEM, measurement of baseline opacity
- treatment by positive and negative control substances and test substance (incubation 4 hour)
- washing epithelium, measurement of opacity after application
-application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C)
- measurement of absorbance (490 nm).

Endpoints measured:
Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quanti
tatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France)
resulting in opacity values measured on a continuous scale.
Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epit
helium on the outer cornea surface through the endothelium on the inner cornea surface) measured in
directly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to
the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while
the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM.
The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC.
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured
with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The
values of absorbance measured at 490 nm were recorded as optical density (OD490) values.

EVALUATION OF RESULTS
Mean opacity:
Opacity values of treated corneas were corrected by subtracting individual background opacity values and
the mean opacity is calculated.

Mean permeability:
Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control
and the mean opacity is calculated.

IVIS calculation:
Resulting mean opacity and OD490 values for each treatment group was combined in an empiricallyderived
formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)

Irritation parameter:

cornea opacity score

Value:

28.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Value:

31.51

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Optical dnesity value

Value:

0.212

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: No prediction can be made on test substance potential to cause eye irritation or serious eye damage.

Conclusions:

The In Vitro Irritancy Score (IVIS) for Reactive Red 45:1 was 31.51. This result could be affected by higher opacity values (colouring of corneas after removing the test substance).
On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.
No prediction can be made on test substance potential to cause eye irritation or serious eye damage.

Executive summary:

The test substance, Reactive Red 45:1,was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 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, Adopted 26^thJuly 2013.

 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured.The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

 

The In Vitro Irritancy Score (IVIS) for Reactive Red 45:1 was 31.51. This result could be affected by higher opacity values (colouring of corneas after removing the test substance).

On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed.

No prediction can be made on test substance potential to cause eye irritation or serious eye damage.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6.9.-8.9.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

The reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) comes from MatTek, Bratislava, SK.
The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS.

Duration of treatment / exposure:

6h

Duration of post- treatment incubation (in vitro):

after removal:
post-soaked-25 min.
post-incubation-18 hours

Number of animals or in vitro replicates:

two replicate tissues

Details on study design:

Temperature: 37±1°C
2 tissues for the test substance
2 tissues for every control
2 tissues as colorant control

Test Procedure
On the day of receipt, EpiOcularTM tissues are conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions.
After pre-incubations, tissues are wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues are topically exposed to the test chemical (50 mg per tissue) for 6 ± 0.25 hours. Four tissues (2 for MTT test 2 for colorant control) are used per test substance (TS) and two for the positive control (PC) and negative control (NC).
At the end of the 6±0.25 hours treatment time, the test articles should be removed by extensively rinsing the tissues with PBS brought to room temperature.
After rinsing, tissues are immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium is intended to remove any test article absorbed into the tissue.
At the end of the post-soak immersion, each insert is removed from the assay medium, the medium is decanted off the tissue, and the insert is blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of warm assay medium. The tissues should be incubated for 18±0.25 hours at standard culture conditions (post-treatment incubation).
At the end of the post-treatment incubation, each insert is removed from the 6-well plate and gently blotted on absorbent material. Two tissues are placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions.
Two tissues undergo the same procedure with medium instead MTT solution (colorant control).
In the end of staining (incubation with medium) the bottom of all inserts is blotted on absorbent material, and inserts are then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol is flowing into the insert. The plates are sealed with parafilm, and are either stored overnight at 2-8°C in the dark or immediately extracted. To extract the MTT, the plates are placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature.
After extraction extracts are collected, mixed and two 200 μl aliquots are transferred to the appropriate wells of a pre-labeled 96-well plate for OD570 reading.

Irritation parameter:

other: viability of treated tissues

Value:

1.98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Table No. 1: OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities.

	Treatment	OD570 of tissues				Avg	SD	Average viability
		Tissue 1		Tissue 2				(% NC)
	water	2.225	2.188	2.362	2.386	2.290	0.084	100.00
NC	%	97.2	95.5	103.1	104.2	100.0	3.66
	273/15	0.049	0.050	0.051	0.052	0.050	0.001	2.20
C2	%	2.1	2.2	2.2	2.3	2.21	0.04
C2CC	273/15-CC	0.003	0.003	0.008	0.007	0.005	0.002	0.23
	%	0.13	0.13	0.35	0.31	0.22	0.10
PC	99% MA	0.412	0.394	0.210	0.205	0.305	0.098	13.33
	%	18.0	17.2	9.2	9.0	13.33	4.27

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance Reactive Red 45:1 was 2.20 % of negative control average value. The measured value was then corrected, due to red colour of the test substance.
Corrected viability of treated tissues was 1.,98 %, i.e. viability was ≤ 60 %.
The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).
According to the classification criteria given in chapter 4.5., the test substance, Reactive Red 45:1, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Further testing with other test methods will be required.

Executive summary:

The test substance, Reactive Red 45:1, was assayed for the in vitro eye irritation in human cornea-like epithelial model EpiOcular^TM.The test was performed according to the OECD Test GuidelineNo.492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissuesurface. Length of exposition was6 hours at 37±1°C in humidified CO₂incubator (5±1% CO₂). Two tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Direct MTT reduction of test substance was excluded in another study.

The assumed colour interference was solved with using of two colorant control tissues incubated by the same way as the other tissues except of colouring in MTT medium. Instead, these tissues were kept in assay medium. Average “viability” of the two tissues was then subtracted from viability of tissues incubated with MTT medium.

Under the above-described experimental designaverage viability of treated tissues was 2.2% (1.98 % after correction),i.e. viability was ≤60 %.

The effect of the test substancewaspositive inEpiOcular^TMmodel (tissues were damaged).

According to the classification criteria given in chapter 4.5., the test substance,Reactive Red 45:1, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Further testing with other test methods will be required.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29.05.-13.06.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted October 2, 2012

Deviations:

yes

Remarks:

In the study only non-opioid analgesic was used for systemic analgesia. This change was performed because of avoiding a negative side effects of opioid analgesic.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Lysolaje, Czech Republic, RČH CZ 21760118
- Weight at study initiation: 2,9-3.0 kg
- Housing: Conventional animal room – individually in metallic cages
- Diet: Pelleted standard diet for experimental animals ad libitum
- Water: Drinking tap water ad libitum (quality corresponding to the Regulation No.: 252/2004 Czech Coll. of Law)
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Room temperature: 20± 3°C, permanently monitored
- Relative humidity: 30 – 70%, permanently monitored
- Light period: 12 hour light/12 hour dark

Study Time Schedule
Animal supply: 24. 05. 2017
Experimental part of study: 29. 05. – 05. 06. 2017
Evaluation of results and final report elaboration: 12. 06. – 26. 06. 2017

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0,1 g
- Test substance: it was used in delivery form
-pH approximately 7

Duration of treatment / exposure:

24h

Observation period (in vivo):

24, 48, and 72 hours

Number of animals or in vitro replicates:

1 female/initial test
2 females/confirmatory test

Details on study design:

TEST PROCEDURE
- 60 minutes before the test substance application Metacam 0.1 mL/kg was administered by subcutaneous injection to provide a therapeutic level of systemic analgesia.
- 5 minutes before the test substance application one or two drops of a topical ocular anesthetics Benoxi 0.4% was applied to each eye.
- 8 hours after the test substance application Metacam 0.1 mL/kg was administered by subcutaneous injection to provide a therapeutic level of systemic analgesia.


PREPARATIONS
- Preparation of experimental animals
Approximately 24 hours before application both eyes of rabbits were examined to remove animals with eye irritation, ocular defects or corneal injury. Only healthy animals without eye defects were used for testing.

- Preparation of the test substance
The test substance was used in delivered form and it was weighted in quantities 0.1g per animal immediately before application.


APPLICATION OF THE TEST SUBSTANCE
- The test substance was placed in the conjunctival sac of one eye of animal after gently pulling the lower lid away from the eyeball.

CLINICAL OBSERVATION
The eyes were examined at 1, 24, 48 and 72 hours after application. After recording the observations at 72 hours, the eyes of rabbit were examined with the aid of fluorescein and the ophthalmoscopy. The grades of ocular reaction for single animal (observation of conjunctivae, cornea and iris) were recorded at each examination. In the end of observation period the animals were sacrificed by injection of veterinary preparation T61 (1 mL/kg).

TOOL USED TO ASSESS SCORE: fluorescein and ophthalmoscopy

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Remarks:

conjunctivae coloured by the test substance

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Remarks:

cornea coloured by the test substance

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Remarks:

conjunctivae coloured by the test substance

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Remarks:

cornea coloured by the test substance

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Remarks:

conjunctivae coloured by the test substance

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

probability of weak irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Evaluation of Results
The following changes were observed on eye at 1 hour after application: conjunctivae – some blood vessels hyperaemic or diffuse, and chemosis – obvious swelling with partial eversion of lids was observed in all rabbits. Some hyperaemic blood vessels of conjunctivae and some swelling above normal in chemosis were observed in all rabbits at 24, 48 and 72 hours after application. All eye lesions disappeared at least at 4th day after treatment.
In all rabbits, the conjunctivae and cornea were coloured by the test substance. The colouring of conjunctivae persisted to the 5th day and in cornea to the 13th day of observation period.
No clinical signs of systemic intoxication were detected.
Examination of eye irritation after single application demonstrated, that the test substance, Reactive Red 45:1, is slightly irritating for eye of rabbit but the injury observed was reversible.

Executive summary:

The test substance, Reactive Red 45:1, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to the OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 2, 2012.

Beforein vivo testing the sequential testing strategyas it is recommended insupplement to TG 405(2012) was respected(see chapter 3. 1.).

The test was performed initially using one animal (No. 1). Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals (No. 3 and No. 5).

 

The following changes were observed on eye at 1 hour after application:conjunctivae –some blood vesselshyperaemicor diffuse, and chemosis –obviousswelling with partial eversion of lidswas observed in all rabbits.Some hyperaemic blood vesselsof conjunctivaeand some swelling above normal in chemosis were observed in all rabbits at 24, 48 and 72 hours after application. All eye lesions disappeared at least at 4^thday after treatment.

In all rabbits, the conjunctivae and cornea were coloured by the test substance. The colouring of conjunctivae persisted to the 5^thday and in cornea to the 13^thday of observation period.

No clinical signs of systemic intoxication were detected.

Examination of eye irritation after single application demonstrated, that the test substance,Reactive Red 45:1,is slightly irritating for eye of rabbit but the injury observed was reversible.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The test item, Reactive Red 45:1, was assayed for the in vitro skin irritation in human epidermal model EpiDerm^TM.The test was performed according to the OECD Test Guideline No.439. In the first experiment too high difference among viability of tissues was observed in range close to cut off value. The experiment was then repeated. In the repeated experiment all test conditions were fulfilled and average viability of treated tissues was54.3% (53. 4 % after correction), i.e. viability was >50 %.

The effect of the test item was negative in EpiDerm^TMmodel (tissues were not damaged). According to the classification criteria given in chapter 4.5., the test item, Reactive Red 45:1, is considered to have no category in regard to skin irritation. This is confirmed in a rather old in vivo study.

The test substance, Reactive Red 45:1, was tested for the evaluation of potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437. The In Vitro Irritancy Score (IVIS) for Reactive Red 45:1 was 31.51. This result could be affected by higher opacity values (colouring of corneas after removing the test substance). On the basis of score (IVIS) given above the classification according to the criteria of the UN GHS could not be performed. No prediction can be made on test substance potential to cause eye irritation or serious eye damage.

Therefore, Reactive Red 45:1 was assayed for the in vitro eye irritation in human cornea-like epithelial model EpiOcular^TM.The test was performed according to the OECD Test Guideline No.492. Viability of treated tissues was 2.2% (1.98 % after correction), i.e. viability was ≤60 %. The effect of the test substance was positive in EpiOcular^TMmodel (tissues were damaged). According to the classification criteria given in chapter 4.5., the test substance, Reactive Red 45:1, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Finally, Reactive Red 45:1, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to the OECD Test Guideline No. 405. Examination of eye irritation after single application demonstrated, that the test substance, Reactive Red 45:1, is slightly irritating for eye of rabbit but the injury observed was reversible.

Justification for classification or non-classification

Based on the available information, Reactive Red 45:1 does not need to be classified as a skin irritant, nor as an eye irritant.