Trisodium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxy-5-nitrobenzenesulphonato(3-)]chromate(3-)

Endpoint summary

Description of key information

Oral rat LD50: 7000 mg/kg bw

Dermal rat LD50: 2500 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Principles of method if other than guideline:

The sample was applied to rats in CMC aqueous solution. After substance administration, the symptoms were observed for 14 days.

GLP compliance:

no

Remarks:

pre GLP

Test type:

standard acute method

Species:

rat

Route of administration:

oral: unspecified

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

Aqueous suspension of 0.5 % CMC

Details on study design:

- Duration of observation period following administration: 14 days.
- Necropsy of survivors performed: yes

Dose descriptor:

LD50

Effect level:

7 000 other: mg/kg

Based on:

test mat.

Clinical signs:

Dyspnoea, apathy, prone position, systemic skin discoloration, urine and feces red coloured, dehydration, poor general condition, initially slight weight loss.

Gross pathology:

Acute cardiac dilatation both sides, acute hyperaemia, mucosa in the glandular stomach astringent, fatty tissue dyed.

Interpretation of results:

other: Not classified according to the CLP Regulation

Conclusions:

LD50 oral was stated at 7000 mg/kg

Executive summary:

Method

The sample was applied to rats in CMC aqueous solution. After substance administration, the symptoms were observed for 14 days.

 

Results

LD50 oral was stated at 7000 mg/kg.

Dyspnoea, apathy, prone position, systemic skin discoloration, urine and feces red coloured, dehydration, poor general condition, initially slight weight loss were observed. Furthermore, the gross analyses revealed acute cardiac dilatation both sides, acute hyperaemia, mucosa in the glandular stomach astringent, fatty tissue dyed.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedures cannot be subsumed under a testing guideline, nevertheless are well documented and scientifically acceptable.

Remarks:

Justification for Read Across is detailed in the endpoint summary.

Principles of method if other than guideline:

The experiment was performed on 40 rats (20 males and 20 females). Substance was administrated by oral intubation at doses of 1670, 2780, 3590 and 4640 mg/kg bw. The surviving animals were killed and autopsied after an observation period of 14 days.

GLP compliance:

no

Remarks:

pre GLP

Test type:

standard acute 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: breeding unit of the testing laboratory
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 160 to 180 g.
- Fasting period before study: the rats were starved during one night before starting the treatment.
- Housing: the males and females were segregated and housed in cages (Type 3) in groups of 5.
- Diet: NAFAG, Gossau SG, rat food, ad libitum.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Humidity: ca. 50 %

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20 % (w/v) in 2% CMC

SUBSTANCE PREPARATION
The test item was weighed into an Erlenmeyer flask on a Mettler balance. Before treatment the suspension was homogeneously dispersed with an Ultra-Turrax and during treatment it was kept stable with a magnetic stirrer.

Doses:

1670, 2780, 3590 and 4640 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females x dose x group

Details on study design:

- Duration of observation period following administration: 14 d
- Frequency of observations and weighing: animals were observed for mortality at 1, 24, and 48 hour and weekly thereafter.
- Necropsy of survivors performed: yes. Animals were killed and autopsied after an observation period of 14 days.

Statistics:

The LD50 was calculated by probit analysis method.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

4 706 mg/kg bw

Based on:

test mat.

95% CL:

3 602 - 6 149

Mortality:

No mortality was noted at the lowest dose, 1/10 and 2/10 animals died in the two subsequent intermediate dose levels respectively and the highest mortality of 5/10 animals was recorded at the highest dose level.

Clinical signs:

Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, curved position, diarrhoea and ruffled fur. Diarrhoea became more accentuated as the dose was increased. The surviving animals had recovered within 9 days.

Gross pathology:

No test substance related gross organ changes were observed.

Dose mg/kg bw	Concentration % in formulation	N. of Animals		Died within
				1 hr.		24 hrs.		48 hrs.		7 days		14 days
		M	F	M	F	M	F	M	F	M	F	M	F
1670	20	5	5	0	0	0	0	0	0	0	0	0	0
2780	20	5	5	0	0	0	0	0	0	0	1	0	1
3590	20	5	5	0	0	0	0	1	1	1	1	1	1
4640	20	5	5	0	0	1	3	1	3	1	3	1	4

Interpretation of results:

other: Not classified according to the CLP Regulation

Conclusions:

The acute oral LD50 of test substance in rats of both sexes observed over a period of 14 days is 4706 (3602 - 6149) mg/kg bw.

Executive summary:

Method

The experiment was performed on 40 rats (20 males and 20 females). Substance was administrated by oral intubation at doses of 1670, 2780, 3590 and 4640 mg/kg bw. The surviving animals were killed and autopsied after an observation period of 14 days.

Result

Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, curved position, diarrhoea and ruffled fur. Diarrhoea became more accentuated as the dose was increased. No mortality was noted at the lowest dose, 1/10 and 2/10 animals died in the two subsequent intermediate dose levels respectively and the highest mortality of 5/10 animals was recorded at the highest dose level.The surviving animals had recovered within 9 days.

The acute oral LD50 of test substance in rats of both sexes observed over a period of 14 days is 4706 (3602 - 6149) mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

7 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Currently, there is only a summary reporting some toxicological test results on Acid Red 357 (AR357); unfortunately the original study reports are no more available. Thus, in order to assess the potential oral acute toxicity of the AR357, the available data on the structural analogue Acid Red 315 (AR315 - Similar Substance 01) were considered.

AR357 and AR315 are both textile, pre-metalized complex dyes 1:2 and they are manufactured for the same use, thus the characteristics of the final product are strictly similar and share the same functionality. Both AR357 and AR315 are chromate pyrazol sodium salts: AR357 is a trisodium, while AR315 is a disodium salt. They share the same structure, except for the fact that AR357 has two sulphonated groups, instead one as AR315. It is expected that this difference has not impact on the toxicological profile.

The typical commercial batches present some differences. The main component AR357 is commonly greater than 55 percent, while in the case of AR315 the main component was stated at 80 %. From this point of view, the major active ingredient concentration of AR315 leads to consider the read-across approach as conservative.

The major difference in the composition is due to the elevated concentration of inorganic salts (i.e. sodium chloride and disodium sulphate) in the AR357, which is a consequence of the salification procedure. In general, the impurities do not impact the experimental results and the endpoint assessment.

Considering that the substances are both salts derived from strong acids (sulphonic acid derivatives), it is expected that they would be completely dissociated and stable in water for the whole test period in that form.

Furthermore, both substances are expected to have a very low bioavailability, based on molecular weight, steric hindrance, polarisation of the whole molecule. Sulphonation of dyes appears to decrease toxicity by enhancing urinary excretion of the dye and its metabolites. The major degree of sulphonation in the AR357 leads to expectation that it would be excreted quicker and easier than the AR315: the introduction of a sulphate function often decreases bioavailability and toxicity to mammals (Worth et al., 2007). Thus, the read across approach taking into account data on AR315 can be considered as a conservative case.

ACUTE TOXICITY - ORAL ROUTE

The available information about AR357 indicates an LD50 for rats of about 7000 mg/kg; dyspnoea, apathy, prone position, systemic skin discoloration, urine and feces red coloured, dehydration, poor general condition and initially slight weight loss were observed. Furthermore, the gross analyses revealed acute cardiac dilatation, acute hyperemia, mucosa in the glandular stomach astringent and fatty tissue dyed (DyStarColours Distribution GmbH, 1975).

Two tests are available on AR315, in which procedures cannot be subsumed under a testing guideline, nevertheless they are well documented and scientifically acceptable. In both cases, five rats per sex were distributed in treated groups dosed by oral gavage; the surviving animals were killed and autopsied after an observation period of 14 days.

In the older study, the test item was suspended at 20 % with carboxymethyl-cellulose 2 % and administered at doses of 1670, 2780, 3590 and 4640 mg/kg bw. Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, curved position, diarrhoea and ruffled fur.

No mortality was noted at the lowest dose, 1/10 and 2/10 animals died in the two subsequent intermediate dose levels respectively and the highest mortality of 5/10 animals was recorded at the highest dose level. The surviving animals had recovered within 9 days. The acute oral LD50 was stated at 4706 (3602 - 6149) mg/kg bw (Huntsman Textile Effects (Germany) GmbH, 1975). In the more recent study, animals were administered with 1670, 2780, 3590 and 4640 mg/kg bw of test substance suspended in polyethylene glycol. Within 2 hours after treatment the rats in all dosage groups showed sedation, dyspnoea, curved position, diarrhoea and ruffled fur. Diarrhoea became more accentuated as the dose was increased. No mortality was noted at the lowest dose, 2 and 6 deaths over 10 animals per dose were noted in the two subsequent intermediate dose levels respectively and the highest mortality of 10 animals was recorded at the highest dose level. The surviving animals had recovered within 9 to 12 days. The acute oral LD50 was determined at 1717 (1271 - 2433) mg/kg bw (Huntsman Textile Effects (Germany) GmbH, 1978).

The reasons of the sensitive difference in the lethal doses recorded for AR315 cannot be easily identify. Apparently both tests were conducted in appropriate manner. The tested substances have in both cases a significant active ingredient concentration (75 and 82 %); the impurities profiles are not detailed, but they are not suspected to impact the toxicological characterization of AR315. The symptoms recorded in both studies appear comparable, as well as the recovery time of surviving animals.

The long experience and the knowledge about the metal-pyrazole complex dyes class give reasons to suspect a low reliability for the outcomes of the more recent study. Unpublished experimental data about several chromate-pyrazole complex dyes have been provided to the Textile Dyes REACH Consortium¹ and none of the substances belonging to this dye category show oral acute toxicity.

Taking into account these considerations, the available lethal dose value on AR357 and the major degree of sulphonation respect to those of AR315, which leads to expect lower bioavailability and toxicity to mammals, AR357 can be considered as a no acutely toxic substance by oral route.

¹Textile Dyes REACH Consortium was established during 2010 by Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD members ) together with other interested parties.

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available neither on AR357, nor on structural analogous.

Nevertheless, because of the physical state and the trade forms of the substance inhalation is not an appropriate route of exposure. Particle size distribution (REACH&Colours Kft, 2014) showed that AR357 is characterized by particles that are expected to remain in the upper respiratory tract, which is characterized by efficacious defence mechanisms able to remove them. The laser diffraction analysis recorded that the 90 percent of particles have a diameter lower than 47 µm, the 50 percent have a diameter lower than 24 µm and the 10 percent have a diameter lower than 10 µm. In particular, only about 3 % of particles have a diameter lower than 4 µm. From this point of view, inhalation route is expected to be an unlikely route of absorption of the substance.

ACUTE TOXICITY - DERMAL ROUTE

Regarding the acute toxicity by dermal route, because of the physical/chemical properties of the substance, the dermal absorption is expected as negligible. The available information reported in a toxicological summary related to the AR357 indicates an LD50 for rats greater than 2500 mg/kg (DyStarColours Distribution GmbH, 1975).

REFERENCE

DyStarColours Distribution GmbH (1975). Ergebnis der gewerbetoxikologischen vorprüfung. Testing laboratory: BASF Aktiengesellschaft. Report no.: XXV. Owner company: DyStarColours Distribution GmbH. Report date: 1976-12-02.

Worth, A. and G. Tier (2007), A Compendium of Case Studies that helped to shape the REACH Guidance on Chemical Categories and Read Across, EUR report no 22481 EN. Worth A & Patlewicz G (Eds).

Justification for classification or non-classification

According to the CLP Regulation (EC 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 about 7000 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).

The dermal LD50 value was established to be higher than 2500 mg/kg, which exceeded the highest CLP limit for classification (dermal acute toxicity category 4: 1000 < ATE ≤ 2000 mg/kg bw).

In conclusion, the test substance is non classified for oral/dermal acute toxicity, according to the CLP Regulation (EC 1272/2008).