Trisodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)][2-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-5-nitrobenzenesulphonato(3-)]chromate(3-)

Endpoint summary

Description of key information

Oral LD50: 6620.9 mg/kg (6098.6 - 7187.9 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

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: Velaz Prague.
- Weight at study initiation: about 150 g.
- Housing: semparated, in plastic polypropylene cages T4 (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 15 g/animal/day.
- Fasting period before study: the day before of the test, animals were not fed.
- Water: CSN 757111 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.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 20 %

Doses:

3981, 5020, 6310, 7943, 10000 mg/kg

No. of animals per sex per dose:

10 rats x dose

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 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 apparatus. 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.

Statistics:

The LD50 is calculated according to the probit method by Blisse.
Data on mortality, the frequency and using the logarithmic dose levels used, were inserted into a computer and analyzed by the PROBIT program, from October 1991 VUOS.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 6 620.9 mg/kg bw

Based on:

test mat.

Mortality:

3981 mg/kg: 0.0 %
5020 mg/kg: 30.0 %
6310 mg/kg: 30.0 %
7943 mg/kg: 70.0 %
10000 mg/kg: 100.0 %

Clinical signs:

other: After 3 hours post-application at dose level of 10.00 g/kg the following symptoms were recorded: - the appearance of skin and hair: stained with excretions. - nutritional status: cachectic. - appearance of visible mucous membranes: in the range of physiol

Other findings:

Autopsies of dead rats administered at dose of 1000 mg/kg revealed:
- External examination of the cadaver
Appearance of hair, skin: hair contaminated with excretions.
Nutritional status: cachectic.
Natural body openings: no macroscopical, patomorphological changes.
Integrity of the body surface: intect.
Postmortality: rigor mortis.

- Organs of head and neck: no macroscopical, patomorphological changes.

- Internal inspection cadaver
Tongue, esophagus, trachea: no macroscopical, patomorphological changes.
Lungs: pink, spongy consistency, airy, without macroscopical, patomorphological changes.
Heart: brownish-red colour, stiffer consistency, without macroscopical, patomorphological changes.

- Abdominal organs
Stomach: acute dilatation, filled with mash content, wall colour brown.
Guts: filled with sparse content, brown-coloured wall
Liver: brownish-red color, stiffer consistency, without macroscopical, patomorphological changes.
Spleen: stiffer consistency, red colour, without macroscopical, patomorphological changes.
Kidney: brownish-red colour, stiffer consistency, without macroscopical, patomorphological changes.
Peritoneum: no macroscopical, patomorphological changes.
Bladder: empty, without macroscopical, patomorphological changes.

- Urine - biochemical tests not established

Symptoms recorded at the lower dose level administered, are analogous to those of the maximum dose.

Overview of the time interval after the onset of various levels of logarithmic dose

Dose mg/kg	Percent mortality
	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	Day 14
	1/2 hr	3 hrs
3981	0	0	0	0	0	0	0	0	0	0	0	0	0	0
5020	0	30	0	0	0	0	0	0	0	0	0	0	0	0
6310	0	20	10	0	0	0	0	0	0	0	0	0	0	0
7943	0	70	0	0	0	0	0	0	0	0	0	0	0	0
10000	0	70	0	0	0	0	0	0	0	0	0	0	0	0

Overview of mortality by sex of experimental rats at different levels

Mortality for each experimental group of tests for acute oral toxicity.

Dose mg/kg	Number of dead animals		Percentage
	Males	Females
3981	0	0	0
5020	1	2	30
6310	0	3	30
7943	4	3	70
10000	5	5	100

Body weight

Dose mg/kg	Males g/kg			Females g/kg
	Start test	End test	Difference	Start test	End test	Difference
3981	166.4	173	+ 6.6	160	168	+ 8
5020	167.6	178.8	+ 11.22	154.4	158.8	+ 44.4
6310	160.8	171.6	+ 10.8	159.6	154.8	- 4.8
7943	156	148.8	- 7.2	163.2	166	- 3.2
1000	162.8	163.2	- 9.6	148.8	148.4	- 0.4

Results

Dose g/kg	Group	Percentage of mortality	Log. Dose	Probits Exp.
3981	10	0.0	0.59999
5020	10	30.0	0.70070	4.476
6310	10	30.0	0.80003	4.476
7943	10	70.0	0.89998	5.524
10000	10	100.0	1.00000

Interpretation of results:

not classified

Remarks:

Migrated information according to the CLP Regulation (EC 1272/2008) Criteria used for interpretation of results: EU

Conclusions:

Oral LD50: 6620.9 mg/kg (6098.6 - 7187.9 mg/kg).

Executive summary:

The acute toxicological characterization of the substance was determined by Acute toxicity test, according to the OECD guideline 401.

Groups of ten rats (5 males and 5 females) were administrated by oral gavage with 3981, 5020, 6310, 7943, 10000 mg/kg of test material. The LD50 was calculated according to the probit method by Blisse.

Conclusion

The LD50 was calculated to be 6.6209 g/kg (6.0986 - 7.1879 g/kg).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

6 620.9 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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guideline.

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:

not classified

Remarks:

Migrated information according to the CLP Regulation (EC 1272/2008) Criteria used for interpretation of results: EU

Conclusions:

LD50 > 5000 mg/kg.

Executive summary:

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.

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

Conclusion

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Additional information

ACUTE TOXICITY - ORAL ROUTE

The acute toxicological characterization of the substance was determined by Acute toxicity test, according to the OECD guideline 401.

Groups of ten rats (5 males and 5 females) were administrated by oral gavage with 3981, 5020, 6310, 7943, 10000 mg/kg of test material. The LD50 was calculated according to the probit method by Blisse and stated at 6620.9 mg/kg (6098.6 - 7187.9 mg/kg) (Synthesia, a.s. - SBU PaB, 1994).

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity studies by inhalation route are available on ABr365.

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 ABr365 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 50 µm, the 50 percent have a diameter lower than 25 µm and the 10 percent have a diameter lower than 10 µm. In particular, only about 2.7 % 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, 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).

Justification for selection of acute toxicity – oral endpoint

Study conducted according to internationally accepted testing guideline.

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 6620.9 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. Nevertheless, based on the physical state and the trade forms of the substance, there are no reasons of concern and the inhalation can be considered as a non appropriate route of exposure.

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).