Dihydrogen (ethyl)[4-[4-[ethyl(3-sulphonatobenzyl)]amino]-2'-sulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, disodium salt

Endpoint summary

Description of key information

Eye Irritation

Eye irritation was tested in study following OECD testing guideline 405 (BASF 1982). That study did not have GLP status. The test material was an aqueous formulation of 38% content . The guideline requires testing 0.1 ml or 0.1g of a substance. In this case, the relative density of 1.48 and the bulk density of 500 kg/m3 indicates that the volume unit of 0.1 ml is the appropriate dose. It corresponds to ca. 50 mg of the pure, solid test substance. In contrast, the amount applied via the aqueous formulation was 38 mg.

This difference in the amount tested is considered negligible because the already dissolved substance represents a more direct exposure and because the findings were clearly below threshold of classification. Regard the criteria for classification and labelling of GHS, the outcome would not have been different if 50 instead of 38 mg would have been tested. This study therefore is suitable as key study.

The substance resulted in transient blue coloration of the eye and in slight reversible redness and swelling of conjunctivae. Due to the intense blue staining, iris and cornea scores could not be determined at the 1h and 24h reading. Examination with fluorescein on day 8 did not show any adverse findings. The slight findings observed for this formulation is attributed to the 7% content of oxalic acid present in the tested formulation.

Another eye irritation study in rabbits is available for a dry commercial product called Neptune Blue BRA concentrate (Hilltop 1979). Based on the name of the substance and its description as a purple solid, it is Acid Blue 9, however no further proof of identity other than the name is available. Accordingly, this study is assigned a validity score of 4. In this study, a 50% concentrate (described as a dark blue liquid) was also tested. The study followed the procedure that later became the OECD guideline 405, but less details on animal housing and obviously test item are contained in the report. Six New Zealand White rabbits were treated with 0.1 ml or 0.096 mg of the substance and the eyes were scored after 24, 48 and 72 hours. At no point in time, there were adverse effects reported.

Skin irritation

The same test material (38% formulation) as used in the eye irritation study (BASF 1982) was also used for the skin irritation test in rabbits (BASF 1982). The procedure of that test was identical to OECD guideline 404 with the exception that the observation was discontinued after 8 days. Animals were treated with 0.5ml (ca 190 mg) of the formulation under an occlusive wrapping for 4h. Then the test item was removed with water. The substance resulted in blue staining of the skin which interfered with scoring for erythema. In two animals, staining resolved within 8 days. In the other animal, staining was still visible after 8 days. Edema were not observed at any observation time points. Readings were done 30 - 60 min after removal of the test patches as well as 24 h, 48 h, 72 h and 8 d after application.

As discussed for the eye irritation study, the applied amount is lower than the amount prescribed in the OECD guideline. However, since there was no indication of skin irritation, this difference in amounts has no impact on the classification and labelling under GHS.

Another skin irritation study in rabbits is available for a dry commercial product called Neptune Blue BRA concentrate (Hilltop 1979). Based on the name of the substance and its description as a purple solid, it is Acid Blue 9, however no further proof of identity other than the name is available. Accordingly, this study is assigned a validity score of 4. In this study, a 50% concentrate (described as a dark blue liquid) was also tested in addition to the pure solid. The study followed the US EPA OPP 81-5 guideline on acute dermal irritation which is a more stringent protocol than the OECD guideline: The exposure duration was 24h and both intact and abraded skin sites were treated. In general, less details on animal housing and obviously the test item are contained in the report. Six New Zealand White rabbits were treated with 0.5g of the substance in an occlusive wrapping and the skin were scored after 24 and 72 hours. Very sliqht erytherna was noted at one intact site and one abraded site at the 24-hour reading of one animal. The five other animals showed no skin reactions. The rabbit with slight skin reactions showed no reactions at intact skin site at the 72- hour reading and very slight erythema was noted at one abraded site. The absence of a skin reaction triggering classification and labelling according to GHS criteria is confirmed in this study.

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

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

8-day observation period

GLP compliance:

no

Specific details on test material used for the study:

38% aqueous formulation, containing also salts and oxalic acid

Species:

rabbit

Strain:

Vienna White

Details on test animals or test system and environmental conditions:

Mean body weights: males 2.8 kg, female 3.64 kg
One animal per cage (type: stainless steel, 40 x 51 cm); animal identification via ear tattoo.
The animals were housed in fully air-conditioned rooms with a room temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm was 12 h light and 12 h dark.
A standardized animal laboratory diet (about 130 g/animal/day) and tap water (about 250 ml/animal/day) were offered.
Acclimatization period: at least 8 days before study start (same housing conditions as during the study).

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated skin of the same animal

Amount / concentration applied:

ca. 0.5 ml (ca. 0.19 g test substance)

Duration of treatment / exposure:

4 hours

Observation period:

8 days

Number of animals:

3

Details on study design:

Clipping of the fur: at least 15 hours before study start
Application area: 2.5 x 2.5 cm
Applicaiton site: upper third of the dorsal and flank region
Removal of the test substance patches: after application with Lutrol and Lutrol/water (1:1)
Readins: 30 - 60 min after removal of the test patches as well as 24 h, 48 h, 72 h and 8 d after application

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24 - 72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Time point:

other: 24-72h

Remarks on result:

not determinable

Remarks:

The substance resulted in blue staining of the skin which interfered with scoring

Irritant / corrosive response data:

Erythema value not readible due to substance-coloured skin. In two animals, staining resolved within 8 days. In the other animal, stiaining was still visible after 8 days.

Interpretation of results:

GHS criteria not met

Conclusions:

An aqueous formulation containing 38% of the substance is not irritating to 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:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

0.1 ml of a 38% aqueous solution were applied. This correspons to 38 mg. For the solid with a bulk density of 500 kg/m3, 0.1 ml would have been 50 mg. Dosed amount slightly lower than required by guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

only observed until day 8, however all findings had resolved.

GLP compliance:

no

Specific details on test material used for the study:

38% aqueous formulation
(contains 47% water, 7% oxalic acid and other salts)

Species:

rabbit

Strain:

Vienna White

Details on test animals or tissues and environmental conditions:

Mean body weights: males 3.0 kg, female 2.91 kg
One animal per cage (type: stainless steel, 40 x 51 cm); animal identification via ear tattoo.
The animals were housed in fully air-conditioned rooms with a room temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm was 12 h light and 12 h dark.
A standardized animal laboratory diet (about 130 g/animal/day) and tap water (about 250 ml/animal/day) were offered.
Acclimatization period: at least 8 days before study start (same housing conditions as during the study).

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye of the same animal

Amount / concentration applied:

0.1 ml (corresponds to 38 mg)

Duration of treatment / exposure:

24 h

Observation period (in vivo):

8 d

Number of animals or in vitro replicates:

3

Details on study design:

Instillation into the conjunctival sac of the right eye.
Assessment: according to OECD and Draize
Readings: 1 h, 24 h, 48 h, 72 h and 8 day after instillation
Body weight determined on day 0 and day 8
Eyes were examined with fluorescein on day 8.

Irritation parameter:

cornea opacity score

Basis:

other: all three animals

Time point:

other: 1h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

cornea opacity score

Basis:

other: all three animals

Time point:

24 h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

cornea opacity score

Basis:

other: all three animals

Time point:

other: 48h, 72h, 8 days

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24 h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

chemosis score

Basis:

other: all three animals

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 48 - 72 h

Score:

0.16

Max. score:

1

Reversibility:

fully reversible within: 72h

Irritation parameter:

iris score

Basis:

other: all three animals

Time point:

other: 1h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

iris score

Basis:

other: all three animals

Time point:

24 h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

iris score

Basis:

animal #1

Time point:

48 h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

iris score

Basis:

animal #2

Time point:

48 h

Remarks on result:

not determinable

Remarks:

The substance is a blue dye, scoring was not possible.

Irritation parameter:

iris score

Basis:

animal #3

Time point:

48 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

other: all three animals

Time point:

other: 72h, 8 days

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 8 days

Irritant / corrosive response data:

Due to transient blue coloring of the eye (for two days in two animals and for one day in one animal), not all values could exactly be determined. The substance is a blue dye.

With the above mentioned restrictions on scoring, no adverse effects were noted on iris and cornea. Swelling of conjunctivae resolved in two animls within 2 days and in one animal within 3 days. Redness of conjunctivae resolved in two animals within 3 days and in one animal within 8 days.

Other effects:

Body weights remained unchanged during treatment
Animal 1: 2.91 / 2.94 kg (day 0 / day 0)
Animal 2: 3.15 / 3.15 kg (day 0 / day 0)
Animal 3: 2.94 / 2.99 kg (day 0 / day 0)

Interpretation of results:

GHS criteria not met

Conclusions:

An aqueuous formulation containing 38% of the substance is not irritating to eyes.

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

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.