6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Endpoint summary

Description of key information

The test substance is not irritating to the skin, but has an irreversible effect on the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Pampaloni s.d.f., Via Pontita 16, 56043 Fauglia, Pisa, Italy
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: approximately 2 kg
- Housing: Individually in stainless steel cages measuring 63 x 48 x 41 cm and equipped with grid floors. Cages were suspended over trays and each tray held an absorbent material which was inspected daily and changed as necessary.
- Diet (e.g. ad libitum): Commercially available anti-biotic free pelleted laboratory diet (Altromin MSK, A. Rieper, Bolzano, Italy), ad libitum
- Water (e.g. ad libitum): Drinking water supplied to each cage via water bottles, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

0.5g

Duration of treatment / exposure:

4 hours

Observation period:

up to 7 days

Number of animals:

3

Details on study design:

On the day of dosing a 0.5 g aliquot of the test substance was mixed to a paste with 2.5 mL of sterile water. This was spread evenly over a 2.5 x 2.5 cm. square of gauze. The gauze was placed on to the animal's skin, with the test substance in contact with the skin. This was covered by a strip of aluminium foil which was held in contact with the skin by encircling the trunk of the animal with an elastic adhesive bandage. All three rabbits of the group were treated in this manner. After four hours the bandages were removed and the treated sites gently cleaned using an absorbent material soaked in warm water.
One hour after removal of the patches and test substance the treated sites were assessed for signs of reaction to treatment. Similar examination was undertaken twenty four, forty eight and seventy two hours and seven days after the end of the four hour dosing period.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 1, 24, 48, 72 hours and 7 days

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1, 24, 48, 72 hours and 7 days

Score:

0

Max. score:

4

Other effects:

Blue staining of the skin.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance has no observable irritant effect on the skin.

Executive summary:

In a GLP compliant skin irritation study, performed according to OECD guideline 404, the test substance (moistened, 0.5g) was applied to the skin of three New Zealand White rabbits for 4 hours. After 4 hours, the treated skin was flushed with water and observation were made for an additional 7 days. The test substance showed a erythema and edema grade of 0 at all observed time points. Under the conditions of this experiment, the test substance was found to be not irritating.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Pampalonl s.d.f., Via Pontita 16, 56043 Fauglia, Pisa, Italy
- Age at study initiation: 9-11 weeks
- Weight at study initiation: approximately 2 kg
- Housing: Indlvldually in stainless steel cages measurlng 63 x 48 x 41 cm and equipped with grid floors. Cages were suspended over trays and each tray held an absorbent material which was inspected daily and changed as necessary.
- Diet (e.g. ad libitum): a commercially available anti-biotic free pelleted Iaboratory diet (Altromin MSK, A. Rieper, Bolzano, Italy) ad libitum
- Water (e.g. ad libitum): Animals were offered drlnking water supplied to each cage via water bottles, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21
- Humidity (%): 45-65
- Air changes (per hr):- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated eye was used as a comparator control.

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

21 days

Observation period (in vivo):

up to 21 days

Number of animals or in vitro replicates:

3

Details on study design:

A 100mg aliquot of the test substance was introduced into the rlght eye of one animal by gently pulling away the lower lid from the eyeball to form a cup into which the test substance was placed. The Iids were then held shut for a few seconds to prevent loss of the test substance. The left eye remained untreated. This animal was dosed as a screen to assess the severity of irritant response. The response to treatment observed was considered not to be unacceptable and did not preclude the treatment of two additional animals, bringing the total to three.
One, twenty four, forty eight, and seventy two hours, seven, fourteen, and twenty one days after dosing the animals were observed under standard conditions and the treated eye examined macroscoptically for damage or irritation to the cornea, iris and conjunctivae' using the untreated eye as a comparator control.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: Average 24, 48 and 72 hours

Score:

1.5

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: Average 24, 48 and 72 hours

Score:

1.8

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

iris score

Basis:

mean

Time point:

other: Average 24, 48 and 72 hours

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 21 days

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: Average 24, 48 and 72 hours

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

Well defined (scores of 2) to moderate (scores of 3) conjunctival irrltation, iris inflammation (score of 1) and areas of corneal opacity (severity score of 2) were apparent in the treated eye of all three anilmals within one hour of dosing. Both the opaque areas of the cornea and the conjunctivae were discoloured blue with the test substance. The sclera and iris also showed blue discolouration, possibly indicating a degree of local absorptlon of the substance.
Corneal opacity and iris inflammation were maintained durlng the first week following dosing. Conjunctival irritation showed some recovery in two animals within forty eight hours, slight reaction being observed at this time. No conjunctival irritatlon remained in any animal after seven days although responses in the cornea and iris remained at this time and one week later, fourteen days after dosing. BIue staining or discolouratlon of aII regions of the eye also remained.
Iris inflammatlon had disappeared within twenty one days of dosing but areas of corneal opaclty were maintained in the treated eye of all animals. At this time, blue colouration was observed in the areas of the cornea showing opaclty, the sclera and conjunctivae. Iridial staining observed earlier in the study showed some reduction in severity but was still present.

Other effects:

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

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance has an irreversible effect on the eye.

Executive summary:

In a GLP compliant eye irritation study, performed according to OECD guideline 405, the test substance (0.1g) was placed in the conjunctival sac of the right eye of three New Zealand White rabbits (RTC 1995). The eyes were examined for 21 days and a mean score was calculated for the observations made at 24, 48, and 72 hours. Scores of 1.5, 1.8, 1, and 2 were observed for conjunctival redness, conjunctival chemosis, iris inflammation, and corneal opacity, respectively. All these effects were fully reversible within 21 days, except for the cornea score.Therefore it was concluded that the test substance had an irreversible effect in the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In a GLP compliant skin irritation study, performed according to OECD guideline 404, the test substance (moistened, 0.5g) was applied to the skin of three New Zealand White rabbits for 4 hours (RTC 1995). After 4 hours, the treated skin was flushed with water and observation were made for an additional 7 days. The test substance showed a erythema and edema grade of 0 at all observed time points. Under the conditions of this experiment, the test substance was found to be not irritating.

Eye irritation:

In a GLP compliant eye irritation study, performed according to OECD guideline 405, the test substance (0.1g) was placed in the conjunctival sac of the right eye of three New Zealand White rabbits (RTC 1995). The eyes were examined for 21 days and a mean score was calculated for the observations made at 24, 48, and 72 hours. Scores of 1.5, 1.8, 1, and 2 were observed for conjunctival redness, conjunctival chemosis, iris inflammation, and corneal opacity, respectively. All these effects were fully reversible within 21 days, except for the cornea score. Therefore it was concluded that the test substance had an irreversible effect in the eye.

Justification for selection of skin irritation / corrosion endpoint:
Only study available

Justification for selection of eye irritation endpoint:
Only study available

Effects on eye irritation: corrosive

Justification for classification or non-classification

Based on the findings in the skin irritation study the test substance does not need to be classified according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 to be irritating to the skin. However, based on the findings in the eye irritation study, the substance need to be classified Xi; R41 according to the Directive 67/548/EEC and Eye damage 1:H318 according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.