Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Endpoint summary

Description of key information

Oral LD50 > 2500 mg/kg bw

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:

February from 3rd to 24th, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. Nevertheless, only the summary of the test report is available, thus many details are missing.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted 17 December 2001

GLP compliance:

yes

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:

Fasted females.

Route of administration:

oral: unspecified

Vehicle:

water

Details on oral exposure:

Test material was administered orally as a suspension in distilled water.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3 female rats per dose

Details on study design:

Clinical signs and body weight development were monitored during the study.
All animals were subjected to gross necropsy.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: A dark brown-coloured liquid discharge from the anus was noted in five animals during the day of dosing. One animal appeared normal throughout the study and the remaining animals appeared normal one day after dosing.

Individual Clinical Observations and Mortality Data

Dose level mg/kg bw	Animal number and sex	Effects Noted After Dosing (h)				Effects Noted During Period After dosing (Days)
		1/2	1	2	4**	1**	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 female	0	0	D*	D*	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2000	1-1 female	0	0	D*	D*	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2000	1-2 female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2000	2-0 female	0	0	D*	D*	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2000	2-1 female	0	0	D*	D*	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2000	2-2 female	0	0	0	D*	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

D* = Dark brown-coloured liquid discharge from the anus

** = Bedding stained dark brown

Individual Bodyweights and Weekly Bodyweight Changes

Dose level mg/kg bw	Animal number and sex	Bodyweight (g) at Day			Bodyweight Gain (g) During Week
		0	7	14	1	2
2000	1-0 female	196	228	236	32	8
2000	1-1 female	206	242	267	36	25
2000	1-2 female	218	256	273	38	17
2000	2-0 female	203	229	240	26	11
2000	2-1 female	205	246	262	41	16
2000	2-2 female	211	231	240	20	9

Individual Necropsy Findings

Dose level mg/kg bw	Animal number and sex	Time of Death	Macroscopic Observations
2000	1-0 female	Killed Day 14	No abnormalities detected
2000	1-1 female	Killed Day 14	No abnormalities detected
2000	1-2 female	Killed Day 14	No abnormalities detected
2000	2-0 female	Killed Day 14	No abnormalities detected
2000	2-1 female	Killed Day 14	No abnormalities detected
2000	2-2 female	Killed Day 14	No abnormalities detected

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 greater than 2500 mg/kg bw.

Executive summary:

Method

The study was performed to assess the acute oral toxicity of the test material following a single oral administration in the Sprague-Dawley CD strain rat. The method followed the OECD Guidelines for the Testing of Chemicals No. 423 "Acute Oral Toxicity - Acute Toxic Class Method". A group of three fasted females was treated with the test material at a dose level of 2000 mg/kg bodyweight. The test material was administered orally as a suspension in distilled water. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

Results

There were no deaths. A dark brown-coloured liquid discharge from the anus was noted in five animals during the day of dosing. One animal appeared normal throughout the study and the remaining animals appeared normal one day after dosing.

Individual body weights and weekly bodyweight changes were normal and individual necropsy revealed no abnormalities.

Conclusion

The acute oral median lethal dose (LD50) of the test material, in the female Sprague-Dawley CD strain rat, was estimated as being greater than 2500 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 500 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

other: read across from supporting substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guideline. Justification for Read Across is detailed in the endpoint summary.

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 bw

No. of animals per sex per dose:

Groups of 6 rats per 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 and 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 bw.

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 administrated 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 bw 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

A good GLP study, performed according to the OECD guideline 423 is available. The study was performed to assess the acute oral toxicity of the Acid Brown 282 (ABr282) following a single oral administration in the Sprague-Dawley CD strain rat. The test material was administered orally as a suspension in distilled water. There were no deaths. A dark brown-coloured liquid discharge from the anus was noted in five animals during the day of dosing. One animal appeared normal throughout the study and the remaining animals appeared normal one day after dosing. Individual body weights and weekly bodyweight changes were normal and individual necropsy revealed no abnormalities (Archroma Germany GmbH, 2005).

Several available studies, in which ABr282 was tested at different purity and at different concentrations, confirm the outcomes of the key study.

ACUTE TOXICITY - INHALATION ROUTE

According to the REACH Regulation Annex VIII Column 2 (specific rules for adaptation from column 1) in addition to the oral route (8.5.1), for substances other than gases, the information mentioned under 8.5.2 to 8.5.3 shall be provided for at least one other route. The choice for the second route will depend on the nature of the substance and the likely route of human exposure. 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, 2012) showed that ABr282 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 115 µm, the 50 percent have a diameter lower than 56 µm and the 10 percent have a diameter lower than 18 µm. In particular, only about 1.44 % 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.

Furthermore, a study by the inhalation route is available. Five male and five female rats were placed in a 435 litre stainless steel chamber with continuous air flow. The test material was introduced into a distilling flask at a constant rate by use of a Harvard pump. The vapours generated were introduced into the air stream. The dosage amount was calculated from the air flow and the amount of liquid material used. The animals were observed hourly during dosing and as late in the day as possible and then once daily for days 1-14. All animals survived and normal body weight changes were noted. All animals were normal at necropsy. Thus, the test material resulted not toxic at a nominal concentration of 0.013 mg/litre (Huntsman Textile Effects (Germany) GmbH, 1979). Nevertheless, the results of the study are not assessable under the CLP Regulation criteria.

ACUTE TOXICITY - DERMAL ROUTE

In the test conducted by dermal route, two male and two female rabbits were dosed at 2.0 g/kg. The test material was applied once on skin to the prepared site under gauze patches and was kept in contact with the skin for 24 hours, at which time the wrappings were removed. Dermal reactions were scored at 25 hours, 7 and 14 days by the Draize scoring system. The LD50 was stated at 2000 mg/kg; the two animals, which died during the study, were observed to have heart and lung abnormalities. These deaths were considered as not treatment related (Huntsman Textile Effects (Germany) GmbH, 1979). Considering the test conclusion, it can not be judged the reliability of the test and the applicability of the results. Furthermore, the substance ABr282 was low in the lot tested and the remaining composition is unknown, thus the study has been disregarded.

A study conducted on the structural analogue 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 administrated 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 and 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 Similar Substance 02 shares with ABr282 the same structure except for the fact that Similar Substance 02 has a sodium nitrobenzenesulphonate group, instead the sodium nitronaphthalene-sulphonate as ABr282 and except for the fact that Similar Substance 02 has one sulphonated group more than ABr282.

Furthermore, the ABr282 is a disodium salt, while Similar Substance 02 is a trisodium salt. Nevertheless, it is expected that these differences have not impact on the acute dermal toxicity.

Justification for selection of acute toxicity – oral endpoint

Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Justification for selection of acute toxicity – inhalation endpoint

Because of the physical state and the trade form of the substance, inhalation is not an appropriate route of exposure.

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 greater than 2500 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).

The dermal LD50 value was established to be higher than 5000 mg/kg body weight, 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).