Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Endpoint summary

Description of key information

An acute oral toxicity test was performed on the test item. The LD50 of the test item was higher than 50 mg/kg and lower than 300 mg/kg b.w. by oral route in rats. In accordance with the OECD guideline No. 423, the LD50 cut-off limit of the test item may be considered as 300 mg/kg body weight by oral route in the rat. According to the CLP criteria the test material was classified as acute toxcity cat. 3. Nevertheless the effects may have been overestimated because of the likely capabilities of the vehicle to enhance absorption of the test item. 
No acute inhalation toxicity test was performed. However, some concerns have been raised in regards to the toxicity per inhalation because of the relatively fine granulometry. Therefore, precautionnary measures should be taken so as to avoid exposition.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2011-02-01 to 2011-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 1 because the study followed the standard guideline of reference (OECD 423), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage JANVIER (53940 Le Genest St Isle - France).
- Age at study initiation: From 8 to 9 weeks.
- Weight at study initiation: Between 172 g and 231 g.
- Fasting period before study: Food was removed one day before the beginning of the study and redistributed 4 hrs after the test item administration.
- Housing: Housed by group of 3 in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid, containing sawdust bedding which was changed at least twice per week.
- Diet : Foodstuff (M20-SDS) ad libitum.
- Water : Tap water ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%):30 to 70 %
- Air changes (per hr): Approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2g (for the first step), 0.3 g (for the second and third step) or 0.05 g (for the fourth and fifth step) of the test item was weighed and 10 mL of DMSO was added. The preparation was magnetically stirred during around 2 days to obtain a dark blue solution.

- Amount of vehicle (if gavage): Administration of 10 mg/kg of body weight.

- Justification for choice of vehicle: the use of distilled water or olive oil gives a suspension. (0.4 g of the test item qsp 2 g with distilled water or olive oil). Because the use of the DMSO gives a homogeous solution (0.4g of the test item qsp 2 g with DMSO), after 1h30 of magnetic stirring, the DMSO was chosen as the vehicule of the study.

Doses:

2000, 300 and 50 mg/kg

No. of animals per sex per dose:

3 female/dose/step
Step 1: 2000 mg/kg b.w.
Step 2: 300 mg/kg b.w.
Step 3: 300 mg/kg b.w.
Step 4: 50 mg/kg b.w.
Step 5: 50 mg/kg b.w.

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Daily examination. Weighed on days D0, D2, D7 and D14.
- Necropsy of survivors performed: yes
- Other examinations performed: Observation and mortality report carried out every day : examination of behavioural or toxic effects on the major physiological functions:
* Body weight
* Clinical signs (spontaneous activity, Preyer's reflex, respiratory rate, convulsions, tremors, body temperature, muscle tone, palpebral opening, mydriasis, salivation, lachrymation, righting reflex, piloerection, mortality)
* Autopsy

Sex:

female

Dose descriptor:

LD50

Effect level:

<= 300 mg/kg bw

Based on:

test mat.

Mortality:

Step 1 at 2000 mg/kg : Death of the 3 rats at 21 hrs 55 min post-dose (1/3) and 45 hrs 50 min post-dose (2/3)
Step 2 at 300 mg/kg: Death of 1 rat on D3 post-dose
Step 3 at 300 mg/kg: Death of the 3 rats at 50 hrs post-dose (2/3) and on D5 post-dose (1/3)
Step 4 and 5 at 50 mg/kg: No mortality occured.

Clinical signs:

other: At 2000 mg/kg (3 rats treated): Decrease in spontaneous activity (3/3), in muscle tone (2/3) and in righting reflex (2/3). Piloerection (3/3). At 300 mg/kg (6 rats treated): Absence or decrease in spontaneous activity (6/6), in body temperature (3/ 6), i

Gross pathology:

At 2000 mg/kg (all dead): The macrosopical examination of the dead animals revealed the presence of a blue liquid in all the digestive system (1/3), the presence of blue aggregrated and/or blue liquid in the stomach (3/3) and a black coloration of liver and spleen (2/3). The marked blue coloration of the stomach prevented from observations.

At 300 mg/kg: The macroscopical examination of the dead animals (4/6) revealed a blue coloration of all the digestive system (4/4), the presence of a blue liquid in the stomach (2/4), thinning of forestomach and corpus (1/4), thickness of the corpus (1/4), and a dark coloration of spleen and liver (1/4). The macroscopical examination of the survival animals (2/6) revealed the presence of white adherences on the forestomach (2/2).

At 50 mg/kg: The macroscopical examination of the animals at the end of the study did not reveal treatment-related changes.

Other findings:

Before the macroscopical examination,
- at 2000 mg/kg, a rigor mortis (3/3) and the presence of a blue liquid on anus (1/3) were noted.
- at 300 mg/kg, a rigor mortis (2/4), blue faeces (1/4) and presence of a blue liquid on anus (1/4) were noted.

Control study TAO-2010 -009 on the Dimethylsulfoxide (DMSO) used as the vehicule in this study

The study was performed from 2010 -12 -14 to 2010 -12 -28 to assess the comportment of the strain of rat used at Phycher laboratory in its environment and to give additional historical data.

The method was designed to meet the requirements of the following:

- OECD guideline for the testing of chemicals N°423

- Method B.1tris of the council regulation N°440/2008

Three animals received the DMSO, administered by gavage under a volume of 10 mL/kg body weight using a suitable syringe graduated fitted with an oesophageal metal canula.

No clinical signs, body weight changes nor treatment related changes were reported (3/3 animals normal)

Interpretation of results:

Toxicity Category III

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The LD50 of the test item Sepisol Fast Violet 3B is higher than 50 mg/kg and lower than 300 mg/kg b.w. by oral route in rats.
In accordance with the OECD guideline No. 423, the LD50 cut-off of the test item may be considered as 300 mg/kg body weight by oral route in the rat.
In accordance with the regulation EC No. 1272/2008 (CLP), the test item must be classified in category 3. The signal word "Danger" and hazard statement H301 "Toxic if swallowed" are required. According to the criteria for classification, packaging and labeling of dangerous substances and preparations in accordance with the E.E.C. Directives 67/548, 2001/59 and 99/45, the test item Sepisol Fast Violet 3B must be classified R22 “Harmful if swallowed”. The item must be characterised by the symbol “Xn” and the warning label “Harmful”.

Executive summary:

The test item Sepisol Fast Violet 3B was administered to a group of 3 females Sprague Dawley rats at the single dose of 2000 mg/kg b.w., then to a group of 6 female Sprague Dawley rats at the single dose of 300 mg/kg b.w., and then to a group of 6 female Sprague Dawley rats at the single dose of 50 mg/kg b.w.

The experimental protocol was established according to the official method as defined in the OECD guideline No. 423 and the test method B.A tris of the Council regulation No. 440/2008.

It was noted the death of the three rats treated at 2000 mg/kg b.w. at 21 hours 55 minutes post-dose (1/3) and at 45 hours 50 minutes (2/3). The mortalities were preceded by a decrease in spontaneous activity (3/3), in muscle tone (2/3) and in righting reflex (2/3), and by piloerection(3/3).

A decrease in the body weight was also noted in animals found dead at 45 hours 50 minutes post-dose: -13% and -17% compared to day 0.

Before the macroscopical examination, a rigor mortis (3/3) and the presence of a blue liquid on anus (1/3) were noted. The macroscopical examination of the dead animals revealed the presence of a blue liquid in all the digestive system (1/3), the presence of blue aggregates and/or blue liquid in the stomach (3/3) and a black coloration of liver and spleen (2/3). The marked blue coloration of the stomach prevented from observations (3/3).

It was noted the death of four rats treated at 300 mg/kg b.w. (4/6): during the second step of the study on day 3 (1/4) and during the third step of the study at 50 hours post-dose (2/4) and on day 5 (1/4). The mortalities were preceded by an absence or a decrease in spontaneous activity (4/4), in Preyer’s reflex (4/4), in body temperature (3/4), in muscle tone (4/4) and in righting reflex (3/4), tremors (1/4), complete or partial ptosis (3/4), piloerection (4/4) and noisy respiration (1/3).

A decrease in the body weight was also noted in animals found dead: -10% to -32% compared to day 0.

Before the macroscopical examination, a rigor mortis (2/4), blue faeces (1/4) and presence of a blue liquid on anus (1/4) were noted. The macroscopical examination of the dead animals revealed a blue coloration of all the digestive system (4/4), the presence of a blue liquid in the stomach (2/4), thinning of forestomach and corpus (1/4), thickness of the corpus (1/4), and a dark coloration of spleen and liver (1/4).

In the survival animals treated at 300 mg/kg b.w. (2/6), a decrease in spontaneous activity (2/2) and in muscle tone (1/2) and piloerection (2/2) were noted on day 1. The animals recovered a normal behavior on day 2.

A decrease in the body weight was noted on day 2 in one animal (1/2): -9% compared to day 0. The animal recovered a normal body weight on day 7.

The macroscopical examination of the animals at the end of the study revealed the presence of white adherences on the forestomach (2/2).

 

No mortality occurred in the animals treated at 50 mg/kg b.w.

No clinical signs related to the administration of the test item treated at 50 mg/kg b.w. were observed. The body weight evolution of the animals treated at 50 mg/kg b.w. remained normal throughout the study. The macroscopical examination of the animals at the end of the study did not reveal treatment-related changes.

 

In conclusion, the LD50 of the test item Sepisol Fast Violet 3B is higher than 50 mg/kg and lower than 300 mg/kg body weight by oral route in rat. In accordance with the OECD guideline N° 423, the LD50 cut-off of the test item may be considered as 300 mg/kg body weight by oral route in the rat.

 

According to the criteria for classification, packaging and labeling of dangerous substances and preparations in accordance with the E.E.C. Directives 67/548, 2001/59 and 99/45, the test item Sepisol Fast Violet 3B must be classified R22 “Harmful if swallowed”. The item must be characterised by the symbol “Xn” and the warning label “Harmful”.

In accordance with the Regulation EC N° 1272/2008 (CLP), the test item must be classified in category 3. The signal word “Danger” and hazard statement H 301 “Toxic if swallowed” are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2011-12-06 to 2011-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 1 because the study followed the standard guideline of reference (OECD 402), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage JANVIER (53940 Le Genest St Isle - FRANCE)
- Age at study initiation: MALE: 8 weeks old. FEMALE: 9 weeks old
- Weight at study initiation: MALE : from 261 g to 290 g. FEMALE: from 220 g to 245 g
- Housing: During the treatment, the animals were kept in individual cages. On D1, the animals were put into their cage by 5, in solid-bottomed clear polycarbonate cages with a stainless steel mesh lid.
- Diet (e.g. ad libitum): ad libitum (foodstuff: M20-SDS)
- Water (e.g. ad libitum): ad libitum (tap water from public distribution system)
- Acclimation period: of at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 dark/12 light

Type of coverage:

occlusive

Vehicle:

other: dimethylsulfoxide

Details on dermal exposure:

TEST SITE
- Area of exposure:dorsal area of the trunk
- % coverage: at least 10% of the body surface cleared for the application of the test item
- Type of wrap if used: porous gauze dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsing with dimethylsulfoxide
- Time after start of exposure: After 24 hours exposure period

TEST MATERIAL and VEHICULE:
- Concentration: 4g or 2g of the test item was weighed and dimethylsulfoxide was added in a 20 mL or 10 mL volumetric flask, respectively. The preparation was magnetically stirred to abtain a blue solution just before administration.
-Administration: The preparation was administered under a volume of 10 mL/kg body weight

Duration of exposure:

Exposure period of 24 hours

Doses:

Group 1: 2000 mg/kg
Group 2: 2000 mg/kg

No. of animals per sex per dose:

Group 1: 5 male rats
Group 2: 5 female rats

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Daily examination (spontaneous activity, preyer's reflex, respiratory rate, convulsions, tremors, body temperature, muscle tone, palpebral opening, pupil appearance, salivation, lachrymation, righting reflex, back hair appearance). Weighing on days D0 (before administration of th test item), D2, D7 and D14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: Macroscopic examinations at the end of the study (after anaesthesia) with the examination of organs likely to be modified in cases of acute toxicity (oesophagus, stomach, the entire digestiv tract, spleen, liver, thymus, trachea, lungs, heart, kidneyx, urinary bladder, testicules, skin of the treatment area, adrenals, pancreas).

Sex:

male

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured during the study

Clinical signs:

other: T0 + 1h, 3h and 5hours: Decreased of spontaneous activity in all treated animals in both groups. D1 to D14: Blue coloration of the treatment site in all treated animals in both groups. D3 to D7: Erythema and dryness at the treatment site in all treated

Gross pathology:

No gross pathology or changes was observed.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The LD50 of the test item Sepisol Fast Violet 3B is higher than 2000 mg/kg body weight by dermal route in the rat in both sex.
According to the criteria for the classification, packaging and labelling of dangerous substances and preparations in accordance with the E.E.C directives 67/548, 2001/59 and 99/45, the test item Sepisol Fast Violet 3B must not be classified. No symbol or risk phrase is required.
In accordance with the Regulation EC n°1272/2008, the test item must not be classified. No signal word or hazard statement is required.

Executive summary:

The test item Sepisol Fast Violet 3B was applied onto the intact skin of 10 Sprague Dawley rats (5 males and 5 females) at the single dose of 2000 mg/kg body weight. The experimental protocol was established on the basis of the official method as defined in the OECD guideline n° 402 dated February 24th, 1987 and the test method B.3. of the Council regulation n°440/2008 of 30 May 2008.

No mortality occured during the study.

A decrease in spontaneous activity was noted in all treated animals (10/10) on day 0. From day 1, no systemic clinical sign related to the administration of the test item was observed. From day 1 to day 14, a blue coloration of the treatment site was noted in all treated animals. Erythema and dryness were noted in all animals from day 3 to day 7.

A decrease in body weight was noted in treated females at 48 hours post-dose, with a mean of -12 % compared to day 0. Then the body weight evolution was normal, comparable with the body weight evolution noted in the historical control group.

The body weight evolution of the males remained normal throughout the study.

The macroscopical examination of the animals at the end of the study did not reveal treatment-related changes.

In conclusion, the LD50 of the test item Sepisol Fast Violet 3B is higher than 2000 mg/kg body weight by dermal route in the rat in both sex.

According to the criteria for the classification, packaging and labelling of dangerous substances and preparations in accordance with the E.E.C directives 67/548, 2001/59 and 99/45, the test item Sepisol Fast Violet 3B must not be classified. No symbol or risk phrase is required.

In accordance with the Regulation EC n°1272/2008, the test item must not be classified. No signal word or hazard statement is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

In the oral acute toxicity assay performed, the test item was solubilized in DMSO. According to the laboratory, DMSO was the only suitable vehicle that could be used to solubilize the test item. It is generally accepted that this solvent may enhance the uptake of substances dissolved in it. Therefore it is likely that an oral absorption of the test item with no solvent or another solvent will lead to a different bioavailability of the test item and a less lethal answer. In the absence of other evidence, our conclusion remains the same: the appropriate classification should be acute category 3.

Sepisol Fast Violet 3B also shows a high Kow value which would suggest a good bioavaibilitity following a penetration through the alveoli. However the substance is a powder and is practically insoluble. As a result, it is unlikey that the substance will be solubilized. In this case the substance would principaly remain as a particle state and the absorption should be very low.

Moreover the vehicle (DMSO) used in the acute toxicity per os is not appropriate for this way of exposure.This would imply a change in the level of bioavailability.

As a result of the previously mentioned reasons, Sepisol Fast Violet 3B is also not expected to be toxic per inhalation but eventualy could be harmful. The precautionary principle would recommend the warning of this possible effect inside safety documents.

Justification for selection of acute toxicity – oral endpoint
Because the study followed the standard guideline of reference (OECD 423), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Justification for selection of acute toxicity – dermal endpoint
Because the study followed the standard guideline of reference (OECD 402), which describes a procedure designed to evaluate this endpoint. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.