Disodium 3-[[4-amino-9,10-dihydro-9,10-dioxo-3-[sulphonato-4-(1,1,3,3-tetramethylbutyl)phenoxy]-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Endpoint summary

Description of key information

Oral LD50 (male and female) > 2000 mg/kg bw

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 December 18, 1990 to January 18, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston, Kent, U.K.
- Age at study initiation: at the start of the main study, rats were approximately five to eight weeks old.
- Weight at study initiation: at the start of the main study, males 170 - 200 g and females 143 - 157 g.
- Fasting period before study: overnight fast immediately before dosing and for approximately two hours after dosing.
- Housing: the animals were housed in groups of up to five by sex in solid-floor polypropylene cages with sawdust bedding.
- Diet: Rat and Mouse Expanded Diet No. l (Special Diet Services Limited) was allowed throughout the study.
- Water: free access to mains drinking water.
- Acclimation period: a minimum period of at Ieast five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 17 - 21 °C. On occasions the temperature was below the Iower Iimit specified in the protocol (19 °C). This did not affect the purpose or integrity of the study.
- Humidity: 42 - 62 %
- Air changes : approximately 15 changes per hour.
- Photoperiod: 12 hours continuous Iight and 12 hours darkness.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

For the purpose of the study the test material was freshly prepared, as required, as a suspension at the appropriate concentration in distilled water.
The composition and stability of the test material and the stability of the preparations were not determined.
- Dose volume: 10 ml/kg
- Concentration: 200 mg/ml

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females

Details on study design:

MAIN EXPERIMENT
- Duration of observation period following administration: 14 days
- Frequency of observations: deaths and overt signs of toxicity were recorded 1/2, 1, 2 and 4 hours after dosing and then daily for 14 days.
- Frequency of weighing: individual bodyweights were recorded on the day of treatment (day 0) and on days 7 and 14 or at death.
- Necropsy of survivors performed: yes; at the end of the study the animals were killed by cervical dislocation. The abdominal and thoracic cavities were opened for examination of all major organs. The macroscopic appearance of any abnormal organs was recorded. No tissues were retained.

DOSE RANGE-FINDING
- Dose levels: 5000, 2000 and 200 mg/kg bw.
- No of animals per dose: 1 male and 1 female.
- Observations: deaths and overt signs of toxicity were recorded 1/2, 1, 2 and 4 hours after dosing and then daily for five days. Individual bodyweights were recorded on the day of dosing to allow calculation of individual treatment volumes. No necropsies were performed.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

MAIN EXPERIMENT
One female was found dead approximately six hours after dosing.

DOSE RANGE-FINDING
One male treated with 5000 mg/kg was found dead one day after treatment.

Clinical signs:

other: MAIN EXPERIMENT Isolated incidents of toxicity noted during the study were hunched posture and lethargy. Purple-coloured staining of the fur was commonly noted during the study. DOSE RANGE-FINDING Signs of toxicity noted were hunched posture, lethargy an

Gross pathology:

MAIN EXPERIMENT
Abnormalities noted at necropsy of the female that died during the study were abnormally red lungs, dark liver and dark kidneys.
No abnormalities were noted at necropsy of animals killed at the end of the study.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

LD50 (male and female) > 2000 mg/kg bw

Executive summary:

The study was performed to assess the acute oral toxicity of the test material in the Sprague-Dawley strain rat. The method used followed the recommendations of the OECD Guidelines for Testing of Chemicals (1981) No. 401 "Acute Oral Toxicity" referenced as Method B1 in Commission Directive 84/449/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Based on the results of the dose range finding experiment, 5 males and 5 females were treated at a dosage level of 2000 mg/kg bw. The substance was administered as a single dose by gavage. After administration of the compound, the animals were observed for 14 days; at the end of the observation period, surviving animals were killed and an autopsy performed.

One female was found dead approximately six hours after dosing; abnormalities noted at necropsy of the female that died during the study were abnormally red lungs, dark liver and dark kidneys.

Isolated incidents of toxicity noted during the study were hunched posture and lethargy. Purple-coloured staining of the fur was commonly noted during the study. Surviving animals showed expected gain in bodyweight during the study. No abnormalities were noted at necropsy of animals killed at the end of the study.

Conlcusion

LD50 (male and female) > 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

ORAL ACUTE TOXICITY

The study was performed to assess the acute oral toxicity of the Acid Violet 048 in the Sprague-Dawley strain rat. The method used followed the recommendations of the OECD Guidelines 401. Based on the results of the dose range finding experiment, 5 males and 5 females were treated at a dosage level of 2000 mg/kg bw. The substance was administered as a single dose by gavage. One female was found dead approximately six hours after dosing; abnormalities noted at necropsy of the female that died during the study were abnormally red lungs, dark liver and dark kidneys. Isolated incidents of toxicity noted during the study were hunched posture and lethargy. Purple-coloured staining of the fur was commonly noted during the study. Surviving animals showed expected gain in bodyweight during the study. No abnormalities were noted at necropsy of animals killed at the end of the study.

ACUTE TOXICTY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on Acid Violet 048. Nevertheless, because of the physical state of the substance inhalation is not an appropriate route of exposure. The vapour pressure of the substance is estimated to be negligible; the particle size distribution showed that the 50 % of particles have a diameter higher than 25 µm; particles having a diameter equal/lower than 4 µm are ca 8 %. Thus, Acid Violet 048 is mainly characterized by particles not-respirable. This consideration, together with the consideration that the substance is manufactured and handled with suitable risk management measures and with the suitable personal protective equipments, lets to consider the possible absorption of the substance by inhalation route as negligible.

DERMAL ACUTE TOXICTY

A study was performed to assess the acute dermal toxicity of Acid Violet 048 in CFY rats. Unfortunately, details about the composition of the tested lot are no more available, thus it cannot be confirm if the test item can be considered as representative of the lot currently in use. The test has been here reported for completeness sake; it has not been considered for classification purposes.

The test item was prepared as a 50 % aqueous solution at the dose level of 5000 mg/kg bw. No deaths occurred. Signs of reaction to treatment consisted of slight lethargy only. No signs of irritation to the skin were seen during the fourteen days observation period. Recovery of all animals, as iudged by external appearance and behaviour, was apparently complete within twenty-four hours of treatment. The bodyweight increases of the treated animals resulted to be normal compared with controls. Autopsy findings were normal.

According to the REACH Regulation, Annex VIII, Column 1 Standard information required, testing by the dermal route does not need to be conducted if the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation).

Furthermore, in the Commission Regulation (EU) 2016/863 it is explained that recent scientific analysis of available data from in vivo acute toxicity studies have shown that substances that are not toxic via the oral route may be expected with high certainty to be also non-toxic via the dermal route. Therefore, testing those substances via the dermal route does not provide essential information for their safety assessment. The amendment is the consequence of studies and scientific debates. In particular, the 15th Meeting of Competent Authorities for REACH and CLP (CARACAL, 2014) concluded that an adaptation of point 8.5.3 of Annex VIII to REACH is justified in order to not require information on acute dermal toxicity for substances that have shown no toxicity in acute oral toxicity test up to the limit dose of 2000 mg/kg bw.

In the oral acute toxicity test on Acid Violet 048 no signs of systemic toxicity were recorded up to 2000 mg/kg bw. The skin sensitisation potential was investigated in Local Lymph Node Assay (LLNA) in mice and no test item-related systemic clinical signs were observed. No reason of concern is raised on the basis of the skin irritation investigation.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.1 Acute toxicity section, substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

The oral LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute oral toxicity (oral acute toxicity category 4: 300 < ATE ≤ 2000 mg/kg bw).

Inhalation exposure is unlikely and the substance is expected to be non harmful/toxic in contact with skin.

In conclusion, Acid Violet 048 does not meet the criteria to be classified for oral acute toxicity, according to the CLP Regulation (EC) No 1272/2008.