Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Endpoint summary

Description of key information

Not skin irritant

Irreversible effects on the eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

(SPF-Quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Source: Broekman Institute, Someren, The Netherlands.
-Age at study initiation: Approx. 12 weeks
-Weight at study initiation: 2528 - 2633 grams
-Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
-Diet: Standard laboratory rabbit diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands) approx. 100 gram per day.
-Water: Free access to tap-water diluted with decalcified water.
-Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21 °C
-Humidity (%): 55 %
-Air changes (per hr): 15 air changes per hour
-Photoperiod (hrs dark / hrs light): 12 hours cycle dark/light with a fluorescent light

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
-Amount applied: 0.5 gram

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): The contralateral flank was similarly prepared (but without test substance and vehicle) to act as a procedural control.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3 male rabbits

Details on study design:

TEST SITE
-Area of exposure: 10x15 cm^2
-Type of wrap if used: Scotchpak-non-woven patch (2x3 cm, 3M, St.Paul, U.S.A.) mounted on Micropore tape (3M, St. Paul, U.S.A.).
The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, U.S.A.).

REMOVAL OF TEST SUBSTANCE
-Washing (if done): no, removed using a tissue moistened with tap-water
-Time after start of exposure: 4h

OBSERVATION TIME POINTS
Viabllity/Mortality/Toxicity: Twice daily.
Body Weight:  Day of treatment (prior to application).
The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance.

SCORING SYSTEM
Erythema and Eschar Formation
No erythema:0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
Edema Formation
No edema: 0
Very slight edema (barely perceptible): 1
Slight edema (edges of area well defined by definite raising): 2
Moderate edema (raised approximately 1 mm): 3
Severe edema (raised more than 1 mm and extending beyond area of exposure): 4

Irritation parameter:

erythema score

Basis:

other: all animals

Time point:

24/48/72 h

Score:

0

Remarks on result:

other: shaved skin

Irritation parameter:

edema score

Basis:

other: all animals

Time point:

24/48/72 h

Score:

0

Remarks on result:

other: shaved skin

Irritant / corrosive response data:

No skin irritation was caused by the test substance after a 4 hour dermal application.
There was no evidence of a corrosive effect on the skin.

Other effects:

Blue staining of the treated skin by the test substance was observed in the treated skin-areas of all animals on day 1.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: Not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

The substance is not irritating for rabbit skin.

Executive summary:

The skin irritation test was performed to rabbit skin according to the OECD Guidelines 404 (1981) and the method B.4 of EEC-Directive 92/69 EEC. The test substance was found to be not irritant when applied to intact rabbit skin: local signs (mean values 24/48/72 hours) consisted of grade 0 erythema and grade 0 oedema. The test substance caused blue staining of the treated skin only on day of the application. No corrosive effects were noted on the treated skin of any animal at any measuring interval.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

(SPF-Quality)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
-Source: Broekman Institute, Someren, The NetherTands.
-Age at study initiation: Approx. 13 weeks
-Weight at study initiation: 2451 - 2802 grams
-Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
-Diet: Standard laboratory rabbit diet (LKK-20, pellet diameter 4 mm, Hope Farms,
Woerden, The Netherlands) approx. 100 g per day.
-Water: Free access to tap-water diluted with decalcified water.
-Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21°C
-Humidity (%): 55 %
-Air changes (per hr): 15 air changes per hour
-Photoperiod (hrs dark / hrs light): 12 hours cycle dark/light by artificial fluorescent light

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

97 ± 1 mg per animal

Duration of treatment / exposure:

one single injection

Observation period (in vivo):

approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

OBSERVATIONS
-Irritation:Immediately after the 24 hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
-Viability / Mortality/ Toxicity: twice daily.
-Body Weight: once, on day of treatment (prior to instiUation).
-Staining of the treated eye was described and recorded at each observation. In case standard lighting was considered Inadequate for observing minor effects, eye examinations were performed using a pocket flash light. In cases of equivoca! results when comparing the treated and untreated eyes, the illustrated guide from the Consumer Product Safety Commission, Washington, D.C. 20207 was used for additional control purposes.

SCORING SYSTEM
CORNEA
-Opacity: degree of density (area most dense taken for reading)
No ulceration or opacity: 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly
visible: 1
Easily discernible translucent area, details of iris slightly obscured: 2
Nacreous areas, no details of iris visible, size of pupil barely discernible: 3
Opaque cornea, iris not discernible through the opacity: 4
-Area of cornea affected
No ulceration or opacity: 0
One quarter or less but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4
IRIS
Normal: 0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection,
any of these or any combination thereof, iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, haemorrhage, gross destruction (any or all of these): 2
CONJUNCTIVAE
-Redness: (refers to palpebral and bulbar conjunctivae, excluding cornea and iris).
Blood vessels normal
Some blood vessels definitely hyperaemic (injected): 1
Diffuse, crimson colour, individual vessels not easily discernible: 2
Diffuse beefy red: 3
-Chemosis: lids and/or nictating membrane
No swelling: 0
Any swelling above normal (includes nictating membrana): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids more than half closed: 4
-Discharge:
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner canthus of
normal animals): 1
Discharge with moistening of the lids and hairs, just adjacent to lids: 2
Discharge with moistening of the lids and hairs, in a considerable area around the eye: 3.

Irritation parameter:

cornea opacity score

Basis:

other: all animals

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

other: all animals

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

1

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Redness was difficult to score, due to black discolouration by the test substance.

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Redness was difficult to score, due to black discolouration by the test substance. At 24 hours grade 2 for eyelids and at 72 hours grade 1 for sclera.

Irritation parameter:

chemosis score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

2

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: Redness was difficult to score, due to black discolouration by the test substance. At 24 grade 1 and at 72 hours grade 2 for sclera.

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: at 24 hours grade 2 for sclera.

Irritation parameter:

chemosis score

Basis:

animal: #2 and #3

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

other: at 48 and at 72 hours grade 1 for sclera.

Irritant / corrosive response data:

-Irritation: slight dulling of normal lustre was also noted on day 1 in all animals, but had disappeared after 24 hours. Treatment of the eyes with 2% fluorescein revealed no corneal epithelial damage in any of the animals, and confirmed the absence of corneal injury after 24 hours. The irritation consisted of redness (maximum grades 1 -2) and chemosis (maximum grade 2) of the conjunctivae and lacrimation. However, conjunctival redness could not be scored on day 1 and was difficult to score at the following observations, due to black discolouration by the test substance. A small amount of discharge was also noted in animals #1 and #2 during the study.
-Corrosion: there was no evidence of ocular corrosion.
-Colouration: black staining of the conjunctival tissues and of the fur on the head and paws of the animals was noted throughout the observation period.

Interpretation of results:

other: Classified for serious eye damage (Category 1) according to the CLP Regulation (EC 1272/2008)

Conclusions:

The test substance produced irreversible effects on the rabbit eye.

Executive summary:

The eye irritation test was performed to the rabbit eye according to OECD Guidelines 405 (1987) and the method B.5 of EEC-Directive 92/69 EEC. The substance was found to cause in all animals slight dulling of normal lustre at 1 hour, disappeared after 24 hours. Treatment of the eyes with 2% fluorescein revealed no corneal epithelial damage in any of the animals, and confirmed the absence of corneal injury after 24 hours. Mean values at 24, 48 and 72 hours for iris and corneal opacity were 0 in all animals. The substance affected the conjunctivae: redness scores (mean values at 24, 48 and 72 hours ) were 0.67 for animal #1 and 1.33 for animals #2 and #3 and chemosis scores (mean values at 24, 48 and 72 hours ) were 1 for all animals. These effects on conjunctival redness and chemosis were reversible within 72 hours (animal #1) and 7 days (animals #2 and #3). However, conjunctival redness could not be scored on day of treatment and was difficult to score at all the following observations, due to black discolouration caused by the test substance. A black staining of the conjunctival tissues of all animals was noted throughout all the observation period and the reversibility could not be verified.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The skin irritation test was performed to rabbit skin according to the OECD Guideline 404 (1981) and the method B.4 of EEC-Directive 92/69 EEC. The test substance was found to be not irritant when applied to intact rabbit skin: local signs (mean values 24/48/72 hours) consisted of grade 0 erythema and grade 0 oedema. The test substance caused blue staining of the treated skin only on day of the application. No corrosive effects were noted on the treated skin of any animal at any measuring interval.

EYE IRRITATION

The eye irritation test was performed to the rabbit eye according to OECD Guidelines 405 (1987) and method B.5 of EEC-Directive 92/69 EEC. A slight dulling of normal lustre was observed in all animals at 1 hour, but it disappeared after 24 hours. Mean values 24/48/72 h for iris and corneal opacity were 0 in all animals. The substance affected the conjunctivae: redness scores (mean values 24/48/72 h) were 0.67 for animal #1 and 1.33 for animals #2 and #3; chemosis scores (mean values 24/48/72 h) were 1 for all animals. These effects on conjunctival redness and chemosis were considered reversible within 72 hours (animal #1) and 7 days (animals #2 and #3). However, conjunctival redness could not be scored on day of treatment and was difficult to score at all the following observations, due to black discolouration caused by the test substance. A black staining of the conjunctival tissues of all animals was noted throughout all the observation period and the reversibility could not be verified within the 7 day-period of the study.

Justification for classification or non-classification

In the CLP Regulation (EC 1272/2008), skin irritation is defined as "the production of reversible damage to the skin following the application of a test substance for up to 4 hours." A single irritant category (Category 2) is defined and, using the results of animal testing, the classification criteria are presented in Annex I: 3.2.2.7.1, Table 3.2.2.

Based on the results of Acute Dermal Irritation/Corrosion study conducted on the test substance, no classification for skin irritation is warranted under the CLP Regulation (EC 1272/2008).

Moreover the CLP Regulation (EC 1272/2008), defines Serious eye damage as “the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application”. A substance that has the potential to seriously damage the eyes is classified in Category 1 (irreversible effects on the eye). On the basis of the results of animal testing, the classification criteria are presented in Annex I: 3.3.2.6.1., Table 3.3.1.

Based on the results of Acute Eye Irritation/Corrosion study conducted, the substance was found to cause a black staining of the conjunctival tissues of all animals throughout all the observation period and the reversibility could not be verified. Therefore, the test substance should be classified in Category 1 due to irreversible effects on the eye, according to the CLP Regulation (EC 1272/2008).