3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethylxanthylium chloride

Endpoint summary

Description of key information

Skin irritation / corrosion: Under the conditions of this study, the test material is not considered to cause corrosion or irritation to the skin.

Eye irritation: Under the conditions of the study, the test material caused irreversible damage to the eyes and requires classification as Category 1.

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:

16 September 2016 to 30 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008 including most recent amendments

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nousan, Notification No 8147

Version / remarks:

2000 including the most recent partial revisions

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: At start of dosing, the animals were between 12 and 24 weeks old
- Weight at study initiation: At start of dosing, body weights were at least 1.5 kg
- Housing: Animals were individually housed in cages with perforated floors (dimensions 67 x 62 x 55 cm) and shelters (dimensions 40 x 32 x 23 cm).
- Diet: Pelleted diet for rabbits approximately 100 grams per day. Hay and wooden sticks were available during the study period.
- Water: ad libitum access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: at least 10 air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: Not reported

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: watery ethanol (50 % v/v)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL. Since the test material did not mix with water, the powdery test material was moistened with watery ethanol (50 % v/v) immediately before application, to ensure close contact with the animal's skin.

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

3; the study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of skin irritation observed in the first animal.

Details on study design:

TEST SITE
- Area of exposure: Flank. Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimetres (10 x 15 cm).
- Type of wrap if used: The test material was applied to the skin of one flank, using a patch of 2 x 3 cm. The patch was mounted on tape which was wrapped around the abdomen and secured with elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the skin cleaned of residual test material using tap water and watery ethanol (50 % v/v) and watery acetone (50 % v/v).
- Time after start of exposure: Four hours after the application

OBSERVATION TIME POINTS
The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded.

SCORING SYSTEM:
To facilitate scoring, treated skin areas were re-clipped and cleaned of residual test material using watery acetone (50% v/v) at least 3 hours before the observations.
- Method of calculation: The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded.

Erythema and Eschar Formation
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beef redness; where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema is given)

Oedema Formation
0 = No oedema
1 = Very slight oedema (barely perceptible)
2 = Slight oedema (edges of area well-defined by definite raising)
3 = Moderate oedema (edges raised approximately 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No skin irritation was observed after 4 hours of exposure to the test material. There was no evidence of a corrosive effect on the skin.

Other effects:

Pink staining of the treated skin by the test material was observed throughout the study period, which hampered the scoring for erythema on Day 1 only (1 hour observation). There was no effect on the conclusion of the study, since sufficient observations were available for interpretation.
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not considered to cause corrosion or irritation to the skin.

Executive summary:

A primary skin irritation/corrosion study was conducted in the rabbit in accordance with the standardised guidelines OECD 404, EU Method B.4, US EPA OPPTS 870.2500 and JMAFF 12 Nousan Notification No 8147 under GLP conditions.

Three rabbits were exposed to 0.5 grams of the test material moistened with 0.5 mL 50 % watery ethanol. The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of skin irritation observed in the first animal. The test material was applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

No skin irritation was observed after 4 hours of exposure to the test material. Pink staining of the treated skin by the test material was observed throughout the study period, which hampered the scoring for erythema on Day 1 only (1 hour observation point). There was no effect on the conclusion of the study, since sufficient observations were available for interpretation.

Under the conditions of this study, the test material is not considered to cause corrosion or irritation 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:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Method according to the Federal Register 38 No. 187 § 1500.42 (eye)

Version / remarks:

1973

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

no

Remarks:

Study pre-dates GLP

Species:

rabbit

Strain:

other: Albino

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Approximately 3 to 4 kg
- Housing: Animals were singly housed in a stainless steel cage without litter
- Diet: Standard pellet feed for rabbits provided ad libitum
- Water: ad libitum
- Acclimation period: >3 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Humidity: 55 ± 10 % (relative)
- Photoperiod: 12 hour light/12 hour dark cycle (07:00 to 19:00)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg

Observation period (in vivo):

Animals were observed for signs of irritation and toxicity 1, 24, 48 and 72 hours after test material instillation and observations were continued for 7 days.

Number of animals or in vitro replicates:

6 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No rinsing occurred

SCORING SYSTEM: Draize scale

CORNEA
Opacity: degree of density (area most dense is taken for reading)
0: No opacity
1: Scattered or diffuse areas of opacity, details of iris clearly visible
2: Easily discernible translucent area, details of iris slightly obscured
3: Opalescent area, no details of iris visible, size of pupil barely discernible
4: Opaque, iris invisible

IRIS
0: Normal
1: Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) still reacting to light (sluggish reaction is positive)
2: No reaction to light, haemorrhage, gross destruction (any of all these or all together)

CONJUNCTIVAE
Redness: (refers to the most severe effect of palpebral and bulbar conjunctivae excluding cornea and iris)
0: Vessels normal
1: Vessels definitely injected above normal
2: More diffuse, deeper crimson red, individual vessels not easily discernible
3: Diffuse beefy red
Chemosis
0: No swelling
1: Any swelling above normal (includes nictating membranes)
2: Obvious swelling with partial eversion of lids.
3: Swelling with lids about half closed
4: Swelling with lids about half closed to completely closed
Discharge
0: No discharge
1: Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)
2: Discharge with moistening of the lids and hairs just adjacent to lids
3: Discharge with moistening of the lids and hairs a considerable area around the eye

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of all animals that could be scored

Time point:

24/48/72 h

Score:

3.9

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

of all animals that could be scored

Time point:

24/48/72 h

Score:

2

Max. score:

2

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

24/48/72 h

Remarks on result:

not determinable

Remarks:

Not possible to evaluate due to very strong swelling of cornea and iris

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of all animals

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

other:

Remarks:

discharge

Basis:

mean

Remarks:

of all animals

Time point:

24/48/72 h

Score:

2.7

Max. score:

3

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

1 hour post application, strong swelling of the conjunctivae with mild to severe oozing/ weeping of the eye was observed. Four rabbits were observed to have slight opacification of the cornea. From 24 hours post application until end of the observation period, the edges of the eyelids were strongly hardened with discharge for all rabbits. Eyes were initially covered in mucous, which later on became purulent and glued together. Any redness caused by the test material could not be determined due to extreme swelling of the eyes. After seven days post application, the observations were essentially the same as the observations after 72 hours.

Table 1: Individual Scores

Time Point (h)	Rabbit number	Evaluation
		Cornea	Iris	Conjunctival redness	Chemosis	Discharge
24	1	*	*	**	4	3
	2	*	*	**	4	3
	3	*	*	**	4	3
	4	*	*	**	4	3
	5	4	2	**	4	3
	6	3	2	**	4	3
48	1	*	*	**	4	3
	2	*	*	**	4	3
	3	4	2	**	4	3
	4	4	2	**	4	2
	5	4	2	**	4	2
	6	4	2	**	4	2
72	1	*	*	**	4	3
	2	*	*	**	4	3
	3	4	2	**	4	3
	4	4	2	**	4	3
	5	4	2	**	4	2
	6	4	2	**	4	1

*Assesment not possible due to staining of the eye as the test material is a red colour

**Not determined due to strong swelling of the cornea and iris

Interpretation of results:

other: Requires classification as Category 1 in accordance with EU classification criteria

Conclusions:

Under the conditions of the study, the test material caused irreversible damage to the eyes and requires classification as Category 1.

Executive summary:

The potential of the test material to cause eye irritation was investigated in the albino rabbit using the German method according to the Federal Register 38 No. 187 § 1500.42 (eye) which is equivalent to the standardised guideline OECD 405.

A single 100 mg quantity of the test material was applied to one eye in each of 6 rabbits and the untreated eye was used as a control. Animals were observed for signs of irritation and toxicity 1, 24, 48 and 72 hours after test material instillation.

1 hour post application, strong swelling of the conjunctivae with mild to severe oozing/ weeping of the eye was observed. Four rabbits were observed to have slight opacification of the cornea. From 24 hours post application until end of the observation period, the edges of the eyelids were strongly hardened with discharge for all rabbits. Eyes were initially covered in mucous, which later on became purulent and glued together. Any redness caused by the test material could not be determined due to extreme swelling of the eyes. After seven days post application, the observations were essentially the same as the observations after 72 hours.

Under the conditions of the study, the test material caused irreversible damage to the eyes and requires classification as Category 1.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

The potential of the test material to cause skin corrosion was assessed in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 bis under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was checked for possible colour interference with the MTT endpoint and for the non-specific reduction of MTT by the test material. No non-specific reduction of MTT by the test material was shown. However, the test material was positive for colour interference and the compound was considered to be incompatible with the test system.

Under the conditions of the study, the test material was not compatible with the test system and no conclusion could be made on the corrosive potential of the test material.

It is considered that further in vitro testing would not offer any further insights into the possible behaviour of the test material; the properties of the material that prevented the in vitro corrosion test being suitable would also apply to the in vitro irritation test and it is therefore concluded that no further in vitro testing is warranted.

A primary skin irritation/corrosion study was conducted in the rabbit in accordance with the standardised guidelines OECD 404, EU Method B.4, US EPA OPPTS 870.2500 and JMAFF 12 Nousan Notification No 8147 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Three rabbits were exposed to 0.5 grams of the test material moistened with 0.5 mL 50 % watery ethanol. The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of skin irritation observed in the first animal. The test material was applied onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

No skin irritation was observed after 4 hours of exposure to the test material. Pink staining of the treated skin by the test material was observed throughout the study period, which hampered the scoring for erythema on Day 1 only (1 hour observation point). There was no effect on the conclusion of the study, since sufficient observations were available for interpretation.

Under the conditions of this study, the test material is not considered to cause corrosion or irritation to the skin.

Eye irritation

The potential of the test material to cause eye irritation was investigated in the albino rabbit using the German method according to the Federal Register 38 No. 187 § 1500.42 (eye) which is equivalent to the standardised guideline OECD 405. The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

A single 100 mg quantity of the test material was applied to one eye in each of 6 rabbits and the untreated eye was used as a control. Animals were observed for signs of irritation and toxicity 1, 24, 48 and 72 hours after test material instillation.

1 hour post application, strong swelling of the conjunctivae with mild to severe oozing/ weeping of the eye was observed. Four rabbits were observed to have slight opacification of the cornea. From 24 hours post application until end of the observation period, the edges of the eyelids were strongly hardened with discharge for all rabbits. Eyes were initially covered in mucous, which later on became purulent and glued together. Any redness caused by the test material could not be determined due to extreme swelling of the eyes. After seven days post application, the observations were essentially the same as the observations after 72 hours.

Under the conditions of the study, the test material caused irreversible damage to the eyes and requires classification as Category 1.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008 the test material is classified as Category 1 for eye damage (H318: Causes serious eye damage).

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion or irritation.