reaction mass of sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium 1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

Not harmful/toxic if swallowed (LD50 (rat, male/female) > 6000 mg/kg)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

other: data on Acid Blue 225_constituent 1

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The experiment was conducted on one of the substance components. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component; however, the impurity profile (which also includes the Acid Blue 225_constituent 2) resulted to be closely similar to that characterizing the substance under assessment (details are given in the document attached to IUCLID section 13.2).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

other: Tif. RAI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: rats (20 males/20 females) were bred under SPF conditions in testing facility breeding unit.
- Age at study initiation: 6 to 7 weeks old.
- Weight at study initiation: 160 to 180 g.
- Fasting period before study: rats were starved during one night before starting the treatment.
- Housing: males and females were segregated and housed in Macrolon cages (Type 3) in groups of 5.
- Diet: food (NAFAG, Gossau SG, rat food), ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Relative humidity: approximately 50 %.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

The substance was suspended at 10 and 30 % with carboxymethyl cellulose 2 %.

Doses:

1000, 3170, 4640 and 6000 mg/kg

No. of animals per sex per dose:

5 males and 5 female per group

Details on study design:

- Duration of observation period following administration: 7 days
- Necropsy of survivors performed: yes

Preliminary study:

None

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 6 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

In the group dosed with 6000 mg/kg 1 male died after 2 hours after substance administration and 1 female died after 24 hours from substance administration. No additional deaths occurred.

Clinical signs:

other: Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, exophthalmus, curved position and ruffled fur. These symptoms became more accentuated as the dose was increased. The surviving animals had recovered within 4 to 6 days

Gross pathology:

No substance related gross organ changes were seen.

Dose mg/kg	Concentration % of formulation	No. of Animals	Died within
			2 hrs		24 hrs		48 hrs		7 days
			males	females	males	females	males	females	males	females
1000	10	5M and 5F	0	0	0	0	0	0	0	0
3170	30	5M and 5F	0	0	0	0	0	0	0	0
4640	30	5M and 5F	0	0	0	0	0	0	0	0
6000	30	5M and 5F	1	0	1	1	1	1	1	1

Interpretation of results:

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

Conclusions:

LD50 (rat, male/female) > 6000 mg/kg

Executive summary:

An acute oral toxicity was determined for test item using Tif. RAI rats (20 males and 20 females). Before administration by oral intubation, the substance was suspended at 10 and 30 % with carboxymethyl cellulose 2 %. The test material was administered at different doses (5 males and 5 females per dose): 1000, 3170, 4640 and 6000 mg/kg.

In the group dosed with 6000 mg/kg 1 male died after 2 hours after substance administration and 1 female died after 24 hours from substance administration. No additional deaths occurred.

Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, exophthalmos, curved position and ruffled fur. These symptoms became more accentuated as the dose was increased. The surviving animals had recovered within 4 to 6 days. They were killed and autopsied after an observation period of 7 days. No substance related gross organ changes were seen.

Conclusion

LD50 (rat, male/female) > 6000 mg/kg

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

ORAL ACUTE TOXICITY

There is no specific data about the oral acute toxicity potential of Acid Blue 225, therefore the available information on Acid Blue 225_constituent 1 has been taken into consideration. It should be noted that the lot tested was characterized by the Acid Blue 225_constituent 1 as main component; however, the impurity profile (which also includes the Acid Blue 225_constituent 2) resulted to be closely similar to that characterizing the substance under assessment. Therefore, the data can be considered as adequate and the approach can be considered as suitable (details are given in the document attached to IUCLID section 13.2).

An acute oral toxicity was determined for test item using Tif. RAI rats (20 males and 20 females). Before administration by oral intubation, the substance was suspended at 10 and 30 % with carboxymethyl cellulose 2 %. The test material was administered at different doses: 1000, 3170, 4640 and 6000 mg/kg.

In the group dosed with 6000 mg/kg 1 male died after 2 hours after substance administration and 1 female died after 24 hours from substance administration. No additional deaths occurred. Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, exophthalmos, curved position and ruffled fur. These symptoms became more accentuated as the dose was increased. The surviving animals had recovered within 4 to 6 days. They were killed and autopsied after an observation period of 7 days. No substance related gross organ changes were seen.

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 6000 mg/kg, 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).