Chromate(3-), [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, sodium

Endpoint summary

Description of key information

Oral LD50: 5792 mg/kg bw (CL 95 %: 3813 - 14980 mg/kg)

Dermal LD50 > 5000 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 February 06 to March 19, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Kleintier farm Madoerin AG, CH-4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 9 to 11 weeks.
- Weight at study initiation: males 201 - 245 g and female 178 - 212 g.
- Fasting period before study: fasted for 12 to 18 hours (access to water was not interrupted). Food was again presented approximately one hour after dosing.
- Housing: groups of five in Makrolon type-3 cages with standard softwood bedding. The cages were cleaned twice weekly during the test period.
- Diet: ad libitum; pelleted standard Kliba 343, Batch 36/85 and 39/86 rat maintenance diet. Food was analysed for contaminants.
- Water: ad libitum; community tap water from Itingen. Water was analysed for contaminants.
- Acclimation period: at least one week under laboratory conditions, after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 40 - 70 %.
- Air changes: air-conditioned with 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark, music/light period.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

TEST ARTICLE PREPARATION
The test article was placed into a glass beaker on a tared Mettler PK 4800 balance, and the vehicle was added.
A weight by volume dilution was prepared using a homogenizer (Ultra-Turrax, Janke and Kunkel, Staüfen, FRG).
Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
The preparation was made immediately prior to dosing.

VEHICLE
4 % solution of CMC, carboxymethylcellulose sodium salt purum (Fluka AG, Buchs / Switzerland in distilled water).

Doses:

Application Volume/kg body weight:
10 ml at 1000 mg/kg
20 ml at 3000 mg/kg
20 ml at 5000 mg/kg
20 ml at 8000 mg/kg

No. of animals per sex per dose:

5 males and 5 females per group

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: mortality/viability were recorded four times during test day 1, and daily during days 2-15. Body weights were recorded at the test days 1 (pre-administration), at the day 8 and the day 15.
- Necropsy of survivors performed: yes. Necropsies were performed by experienced prosectors. All animals were necropsied. All animals surviving to the end of the observation period were killed by intraperitoneal injection of sodium pentobarbitone.
- Other examinations performed: each animal was examined for changes in appearance and behaviour four times during day 1, and daily during days 2-15.All abnormalities were recorded.

Statistics:

The LOGIT-Model (COX, Analysis of Binary Data, London 1977) was applied to estimate the toxicity value. Additionally, the 90, 95 and 99 % confidence intervals for the toxicity and the slope of the concentration response line were estimated.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

5 792 mg/kg bw

Based on:

test mat.

95% CL:

3 813 - 14 980

Mortality:

1000 mg/kg: no deaths occurred.
3000 mg/kg: one female dead at day 2 and two females dead at the day 3; no males dead.
5000 mg/kg: two females dead at day 2 and one at the day 3; no males dead.
8000 mg/kg: three females dead at day 2 and one at the day 3; two males dead at day 2 and one at the day 3.

Clinical signs:

other: The following symptoms were observed: 1000 mg/kg: sedation, dyspnea, curved body position, ruffled fur. 3000 mg/kg: sedation, dyspnea, ataxia, curved body position, diarrhea, ruffled fur. 5000 mg/kg: sedation, dyspnea, ataxia, curved body position, diarrh

Gross pathology:

1000 mg/kg killed:
lung: partly red, focal (2), partly red, maculate (1), dark-red, mottled (3), no pathologic changes (4)
3000 mg/kg dead:
lung: black, mottled (1), reddish foci/4 mm (1)
liver: black discoloured (1), black (2)
stomach/intestines : black (3)
anal region: black (1)
3000 mg/kg killed:
lung: mottled (3)
no pathologic changes (4)
5000 mg/kg dead
lung: mottled (1)
stomach/intestines : black (2), black contents (1)
5000 mg/kg killed
no pathologic changes (7)
8000 mg/kg dead
liver: black discoloured (7)
lung : mottled (5)
stomach/intestines: black (7)
8000 mg/kg killed
no pathologic changes (3)

Interpretation of results:

other: Not classified as harmful or toxic according to the CLP Regulation (EC) No. 1272/2008.

Conclusions:

LD50: 5792 mg/kg bw (CL 95 %: 3813 - 14980 mg/kg).

Executive summary:

Method

The test article was administered to rats of both sexes by oral gavage, at doses from 1000 to 8000 mg/kg. The study procedures followed fulfil the OECD guideline 401 and EU method B1.

Results

The following death rate was observed:

0 % at 1000 mg/kg;

30 %. at 3000 mg/kg;

30 % at 5000 mg/kg;

70 % at 8000 mg/kg.

Based on these observations, the Logit-Estimation for the acute oral toxicity in rats of both sexes observed for a period of 15 days is 5792 mg/kg with 95 % CI of males/females 3813 - 14980 mg/kg

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 792 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:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Velaz Prague.
- Weight at study initiation: about 200 g (range 200-270 g).
- Housing: individually, in plastic polypropylene cages T3 (supplied by s.p. Velaz Prague).
- Diet: animals were fed with commercial granular food mixture Altromin 1320, supplied by s.p. Velaz Prague. Daily dose of 20 g/animal/day.
- Fasting period before study: the day before of the test, animals were not fed.
- Water: CSN 767111 ad libitum.
- Bedding: wood shavings, from light wood.
- Acclimation period: 1 week.
Cleaning and disinfection of premises menagerie were made at dates determined, according to the standard operating procedures and compliance regime measures.

ENVIRONMENTAL CONDITIONS
- Temperature: controlled temperature at 22 ± 3 °C.
- Humidity: 50 ± 15 %.
- Photoperiod: 12 hrs dark / 12 hrs light, by fluorescent lamp.

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: during one week were twice shaved in the back, for an area of 6 x 6 cm.
- Type of wrap if used: gauze soaked with acetone ethyl alcohol in ratio 1:1 and gauze soaked with physiological solution. Treated skin was covered with gauze, aluminium foil and fixed by technical tape applied to the circumference of the trunk; this was accompanied by a fixation bandage. The dressing was covered with technical tapes and attached around the circumference of the trunk, in order to maintain the test substance in contact with the skin and in order to avoid swallowing of the substance.

TEST MATERIAL
- Solution: weighed sample was added to water and mixing with a metal spatula, to form an aqueous paste.

Duration of exposure:

24 hours

Doses:

5020 mg/kg

No. of animals per sex per dose:

Groups of 6 rats x dose (one group as control).

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: immediately before the substance application, rats were weighed. Immediately after application, ca 30 minutes, after 3 hours post-application. The day after the application the observations were made in the morning and in the afternoon, while in the following days at least once daily. Body weight was measured at the beginning and end of the experiment.
- Necropsy of survivors performed: yes; internal organs were assessed for colour, size, consistency and structure.
- Other examinations performed: appearance of the skin, hair, visible mucous membranes status, mental activity, somatomotor activity, reactivity to stimulus, lacrimation, respiration, digestion, urogenital and circulatory systems. Organs and muscles were examined macroscopically. If post-mortem the bladder of animals was full, the urines were analyzed focusing on the detection of proteins, blood sugar, ketones, bilirubin, urobilinogen and pH.

Sex:

male

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Clinical signs:

other: Throughout the duration of the experiment, no signs of intoxication were observed. Visual examination did not reveal differences in hair, skin, mucous membranes.

Gross pathology:

Hair, skin, mucous membranes: normal.
Subcutaneous and muscles: without macroscopical patomorphological changes.

Other findings:

Results after autopsy:
animals appeared in a good status, with normal motorial activity, reactivity and sensibility.
functionality of digestive, urogenital and circulatory systems appeared normal.
head and neck: with normal motorial activity, reactivity and sensibility.
lung: pink colour, spongy consistency, ventilation without macroscopic pathomorphological changes.
stomach: full of food, without macroscopic pathomorphological changes.
guts: filled with sparse mushy food, without macroscopic pathomorphological changes.
liver: dark reddish brown colour, smooth surface, stiffer consistency, without macroscopic pathomorphological changes.
spleen: red colour, stiffer consistency, without macroscopic pathomorphological changes.
kidney: brownish-red colour, surface smooth, stiffer consistency, without macroscopic pathomorphological changes.
bladder: without macroscopic pathomorphological changes.

Interpretation of results:

other: Not classified as harmful or toxic according to the CLP Regulation (EC) No. 1272/2008.

Conclusions:

LD50 > 5000 mg/kg.

Executive summary:

Method

The acute toxicological characterization of the substance was determined by Acute toxicity test, according to the OECD guideline 402.

Two groups of six rats (one dosed and one used as control) were administered with 5020 mg/kg of test material. Treated skin was covered with gauze, aluminium foil and fixed by technical tape applied to the circumference of the trunk; this was accompanied by a fixation bandage. The dressing was covered with technical tapes and attached around the circumference of the trunk, in order to maintain the test substance in contact with the skin and in order to avoid swallowing of the substance.

Test substance was removed after 24 hours and the observation period was of 14 days.

Results

The LD50 resulted greater than 5000 mg/kg by dermal application.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Additional information

ACUTE TOXICITY - ORAL ROUTE

The oral acute toxicity of Acid Brown 355 was assessed in two studies on rats.

In the key study the test item was administered to rats of both sexes by oral gavage, at doses from 1000 to 8000 mg/kg. The study procedures followed fulfil the OECD guideline 401 and EU method B1. Sedation, dyspnea, ataxia, curved body position, emaciation (males), diarrhea, ruffled fur are the symptoms recorded at the higher dose administered. The surviving rats in all the groups tested had recovered within 2 to 15 observation days. The following death rate was observed: 0 % at 1000 mg/kg; 30 %. at 3000 mg/kg and at 5000 mg/kg; 70 % at 8000 mg/kg. The acute oral toxicity in rats of both sexes observed for a period of 15 days was established at 5792 mg/kg with 95 % CI of males/females 3813 - 14980 mg/kg (Huntsman Textile Effects (Germany) GmbH, 1986).

The test was performed on the Acid Brown 355b (ABr355b - EC 280-689-4), trisodium salt: chromate(3-),[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)][3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)]-, trisodium.

The reference substance under registration is the Acid Brown 355 (ABr355 - EC 284-915-2), the form which can have a variable number of the sodium atoms. Thus, although it has been appointed a different EC number to the ABr355b trisodium salt form (EC 280-689-4), it can be considered as “included” into the structure variability of the ABr355 (EC 284-915-2). In this context, the use of the experimental data available on the ABr355b (EC 280-689-4), in order to assess the ABr355 (EC 284-915-2), can be considered as not a read across, but an evaluation on one of the possible forms of the ABr355.

Furthermore, based on the experience in the manufacture of the AB355, the most frequent form is the trisodium salt.

The supporting study available confirmed the outcome of the key one: the LD50 was identified at 5890 (48763 to 7134) mg/kg. At the 3th day diarrhea effect was observed. Rats die within 2-4 days after application. Autopsy found mild pulmonary congestion. In rats surviving and killed after 14 days, undetected for organs macroscopically observable changes (Synthesia, a.s. - SBU PaB, 1987).

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on ABr355.

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, 2013) showed that ABr355 is characterized by particle 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 79 µm, the 50 percent have a diameter lower than 41 µm and the 10 percent have a diameter lower than 14 µm. In particular, only about 2 % 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 there is not a complete study report of the test conducted on the ABr355: only a summary is available. Test material was administered to rats as an aqueous paste at a dose of 5000 mg/kg and the LD50 was reported as greater than 5000 mg/kg. After application the animals tested showed no signs of intoxication and no death occurred (Synthesia, a.s. - SBU PaB, 1987).

A study conducted on the structural analogue Acid Brown 365 (ABr365 - Similar Substance 02) is available. The substance was assayed in a toxicity test, according to the OECD guideline 402. Two groups of six rats (one dosed and one used as control) were administered with 5020 mg/kg of test material. Treated skin was covered with gauze, aluminium foil and fixed by technical tape applied to the circumference of the trunk; this was accompanied by a fixation bandage. Test substance was removed after 24 hours and the observation period was of 14 days. Throughout the duration of the experiment, no signs of intoxication were observed. Visual examination did not reveal differences in hair, skin, mucous membranes. Subcutaneous and muscles examination showed no microscopic pathomorphological changes. Animals appeared in a good status, with normal motorial activity, reactivity and sensibility. Functionality of digestive, urogenital and circulatory systems appeared normal. Therefore, the LD50 was stated as greater than 5000 mg/kg by dermal application (Synthesia, a.s.- SBU PaB, 1994).

The read across can be considered as representative because ABr365 shares with ABr355 the same structure except for the fact that ABr365 has a sodium nitrobenzenesulphonate group, instead the sodium nitronaphthalene-sulphonate as ABr355. Furthermore, the ABr355 may have a variable number of sodium salts, while ABr365 is a trisodium salt. Nevertheless, it is expected that these differences have not significant impact on the acute dermal toxicity. There are no relevant differences between the tipycal composition of ABr355 and the composition of the ABr365 lot tested.

The read across approach has been further detailed in the report attached to the IUCLID section 13.

Justification for selection of acute toxicity – oral endpoint

Test conducted according to internationally accepted testing guidelines.

Justification for selection of acute toxicity – dermal endpoint

Study conducted according to internationally accepted testing guideline.

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 5792 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).

No data about acute toxicity by inhalation route is available.

The dermal LD50 value was established to be 5000 mg/kg body weight, therefore the test substance is out of any classification limit for acute dermal toxicity (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).