Disodium ar,ar'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[(4-chlorophenoxy)benzenesulphonate]

Endpoint summary

Description of key information

Oral LD50 (female) > 2000 mg/kg body weight

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 May 25 to June 15, 2018

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 December 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: iIn-house bred animals.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 9 to 10 weeks.
- Weight at study initiation: 160.64 to 167.67 g
- Fasting period before study: animals were fasted overnight (16 to 18 hours) prior to dosing; ater was provided ad libitum during fasting period.
- Housing: animals were housed in standard polypropylene cage (430 x 285 x 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube; three per cage.
- Diet: Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided, ad libitum.
- Water: ad libitum.
- Acclimation period: healthy young adult animals used for Step-I and Step-I confirmation were acclimatized for five and seven days respectively to laboratory condition prior to treatment and were observed for clinical signs once daily.
- Health check: veterinary examination of all the animals was performed on the day of receipt and on 5th day of acclimatization.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.6 to 22.9 °C
- Humidity: 43 % to 6 5%
- Air changes:12 to 15 air changes per hour.
- Photoperiod: 12 hours fluorescent light and 12 hours dark cycle.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dose formulations was calculated by the active ingredient content.
- Concentration: 200 (mg/ml)
- Dose volume: 10 ml/kg body weight

Doses:

2000 mg a.i./kg body weight (corresponding to 2520 mg/kg body weight)

No. of animals per sex per dose:

3 female animals per step

Details on study design:

- Study Design: the animals were dosed in a stepwise procedure with three female animals per step. Since no clinical signs of toxicity and mortality was observed at 2000 mg/kg body weight in Step-I, the sequential Step-I confirmation was conducted using three more female rats treated at the same dose level.
- Duration of observation period following administration: 14 days
- Frequency of observations: all the animals were observed for clinical signs of toxicity and mortality at 30 to 40 min, 1 hr (±10 mins), 2 hrs (±10 mins), 3 hrs (±10 mins) and 4 hrs (±10 mins) post dosing on day 1 and once daily thereafter for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. Observations included changes in skin, fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern.
- Frequency of weighing: individual animal body weight was recorded at receipt, on day 1 (before test item administration) and on day 8 and 15 during the observation period.
- Necropsy of survivors performed: at the end of observation period, all the animals were sacrificed under carbon dioxide anaesthesia, subjected to necropsy and a complete gross pathological examination.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No deaths occurred.

Clinical signs:

other: No clinical signs of toxicity and mortality was observed in the animals dosed with 2000 mg/kg body weight in both Step-I and Step-I confirmation.

Gross pathology:

No gross pathological changes were observed in any of the animals dosed at 2000 mg/kg body weight in both Step-I and Step-I confirmation.

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) 1272/2008

Conclusions:

LD50 (female) > 2000 mg/kg body weight

Executive summary:

The test item was evaluated for acute oral toxicity in Sprague Dawley rats as per OECD Guideline No. 423.

A starting dose of 2000 mg/kg body weight was selected.

A total of 09 females (3 females each for each Step-I and Step-I confirmation) were used for the experiment. All the animals of Step-I and  Step-I confirmation were administered with 2000 mg/kg body weight of the test item through oral route.

All the animals were observed for clinical signs of toxicity and mortality at 30 to 40 min, 1 hr, 2 hrs, 3 hrs and 4 hrs post dosing on day 1 and once daily thereafter for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. Body weight was recorded on day 1 before test item administration and on day 8 and 15 during the observation period. At the end of observation period, all the surviving animals were sacrificed under carbon dioxide anaesthesia, subjected to necropsy and gross pathology examination.

In Step-I and Step-I confirmation, the animals dosed with 2000 mg/kg body weight did not reveal any clinical signs of toxicity and mortality. No changes were observed in body weight and percent change in body weight with respect to day 1 at 2000 mg/kg body weight. All the animals revealed physiologically normal increase in the body weight.

No gross pathological changes were observed in any of the animals dosed at 2000 mg/kg body weight.

Conclusion

LD50 (female) > 2000 mg/kg body weight

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Additional information

ACUTE TOXICITY - ORAL ROUTE

The test item was evaluated for acute oral toxicity in Sprague Dawley rats as per OECD Guideline No. 423. A starting dose of 2000 mg/kg body weight was selected. 3 females each for each Step-I and Step-I confirmation were used for the experiment. All the animals of Step-I and  Step-I confirmation were administered with 2000 mg/kg body weight of the test item through oral route.

In Step-I and Step-I confirmation, the animals dosed with 2000 mg/kg body weight did not reveal any clinical signs of toxicity and mortality. No changes were observed in body weight and percent change in body weight with respect to day 1 at 2000 mg/kg body weight. All the animals revealed physiologically normal increase in the body weight. No gross pathological changes were observed in any of the animals dosed at 2000 mg/kg body weight.

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on Acid Green 040.

Nevertheless, because of the physical state of the substance inhalation is not expected to be an appropriate route of exposure. Particle size distribution showed that the ca 92 % of particles has a diameter greater than 45 μm. Thus, most of the Acid Green 040 particles are expected to remain in the upper respiratory tract, which is characterized by efficacious defence mechanisms able to remove them; therefore, respirable particles, able to enter the deepest part of the lung, the non-ciliated alveoli (i.e. D50 of 4 μm), can be considered a minimal portion.

ACUTE TOXICITY - DERMAL ROUTE

Because of the physical/chemical properties of the substance, also the dermal absorption is expected as negligible.

According to the Commission Regulation (EU) 2016/863, amending Annexes VII and VIII to REACH Regulation (EC) 1907/2006, testing by the dermal route does not need to be conducted if no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation). Furthermore, it is explained that recent scientific analysis of available data from in vivo acute toxicity studies has 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.

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 information on acute dermal toxicity can be avoided for substances that have shown no toxicity in acute oral toxicity test up to the limit dose of 2000 mg/kg bw.

In the case of Acid Orange 040, no signs of systemic impairments were found during the in vivo acute oral toxicity test; in addition, the substance resulted to be non skin irritating in rabbits and it can be considered as non-skin sensitising, based on the available data on the structural analogous (the read across approach can be considered as appropriate and suitable; details about the approach are reported into the related skin sensitisation endpoint record).

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).

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