Reaction mass of trisodium 4-amino-3-[[4-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and trisodium 4-amino-3-[[4-[[4-[(4-amino-2-hydroxyphenyl)azo]phenyl]amino]-3-sulphonatophenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

key study

Study period:

From September 27th to October 18th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The test was conducted by means of Read Across approach. The reliability of the source study report is 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted February 24t, 1987

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168:1 - Similar Substance 01

IUPAC Name:

Direct Black 168:1 - Similar Substance 01

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 8 weeks males; 10 weeks females.
- Weight at study initiation: 191 - 206 g males; 168 - 181 g females.
- Fasting period before study: 12 - 18 hours before test administration. Access to water was not interrupted. 2-3 hours after dosing food was again presented.
- Housing: groups of five in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 343, rat maintenance diet, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: one week under laboratory conditions, after veterinary examination.

HUSBANDRY CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12hrs dark / 12 hrs artifical fluorescent light.
- Other: room was airconditioned, music during the light period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled

Details on oral exposure:

APPLICATION VOLUME/Kg bw
10 ml at 2000 mg/kg

PREPARATION
The test article was placed into a glass beaker on a tared balance and the vehicle was added. A weight by volume dilution was prepared using a homogenizer. Homogeneity of the test article in the vehicle maintained during treatment using a magentic stirrer.
The preparation was made immediately prior to dosing.

Doses:

10 ml at 2000 mg/kg b.w.

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days.
- Clinical signs observed: 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. The animals were checked for the following clinical signs: aggressiveness, vocalization, restlessness / excitation, nervousness, fear, sedation, somnolence, sleep, coma, rhinorrhea, epistaxis, apnea, dyspnea, rales, chromodacryorrhea, exophthalmos, miosis, mydriasis, whitish discharge, lid adhesion, lacrimation, negative corneal reflex, akinesia, ataxia, dropped head, hyperkinesia, hypokinesia, paralysis, flaccid, paralysis, spastic, paddling movements, stiff gait, rolling movements, ventral body position, latero-abdominal position, hunched posture, spasms, tonic muscle spasms, clonic muscle spasms, opisthotonus, saltatory, spasms, trismus, tremor, muscle-twitching emaciation and muscle-twitching generalized, erythema, edema, necrosis, loss of weight, diarrhea, ruffled fur, salivation, pallor, cyanosis.
- Frequency of observations: mortality/viability were recorded four times during test day 1(at 1, 2, 3 and 5 hours) and daily during days 2 - 15.
- Frequency of weighing: test day 1 (pre-administration), day 8 and 15.
- Necropsy: necropsies were performed by experienced prosectors. All animals were necropsied. All the animals which survived until the scheduled termination were euthanized by intraperitoneal injection of sodium pentobarbitone.

Statistics:

The LOGIT-Model could not be applied to the observed rates of death. The toxicity was estimated without the use of a statistical model.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred up to 15 days post-treatment.

Clinical signs:

2000 mg/kg: males/females exhibited ruffled fur, diarrhea, hunched posture. Males exhibited sedated.
All animals had recovered after 10 observation days.

Body weight:

The body weight gain of the animals was not affected by the test article treatment throughout the entire study period.

Gross pathology:

No obvious macroscopical organ findings were seen in any animal.

Any other information on results incl. tables

Clinical signs

Summary

Test day	1	1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Time after treatment (hrs)	1	2	3	5
Males
Behaviour sedated	-	-	-	x	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Hunched posture	x	x	x	x	x	x	x	x	x	-	-	-	-	-	-	-	-	-
Ruffled fur	x	x	x	x	x	x	x	x	x	x	x	x	x	-	-	-	-	-
Diarrhea	-	x	x	x	x	-	-	-	-	-	-	-	-	-	-	-	-	-
Females
Hunched posture	-	-	-	x	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Ruffled fur	-	x	x	x	x	x	x	x	x	x	x	x	x	-	-	-	-	-
Diarrhea	-	x	x	x	x	-	-	-	-	-	-	-	-	-	-	-	-	-

X = sign observed

- = observation performed, sign not observed

Test day			1	1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Time after treatment (hrs)			1	2	3	5
Animal N.	Sign	(G)
1M	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
2M	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
3M	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	1	1	1	1	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
4M	Behaviour sedated	(3)	-	-	-	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Hunched posture	(1)	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-	-	-
	Ruffled fur	(3)	1	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
5M	Ruffled fur	(3)	-	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
6F	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
7F	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
8F	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	1	1	1	1	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
9F	Hunched posture	(1)	-	-	-	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-
	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-
10F	Ruffled fur	(3)	-	1	1	1	1	1	1	1	1	1	1	-	-	-	-	-	-	-
	Diarrhea	(1)	-	1	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-

- = observation performed, sign not observed

(G) = max. grade

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

LD50 (males and female) > 2000 mg/kg bw

Executive summary:

The substance was assessed for its acute toxicity to rats in a limit test, according to the OECD guideline 401 and the EU Method B.1. The acqueous solution of the substance was administrated by oral gavage to five female and five male Wistar rats in a concentration of 2000 mg/kg bw. The rats were observed for mortality, clinical signs, macroscopic changes and vitality four times during test day 1 (at 1, 2, 3 and 5 hrs) and daily during days 2 - 15. The animals were weighed on test day 1 (pre-administration), day 8 and 15.

During the observation period, no mortality was observed and no macroscopical findings were noted. The body weight was increased. Clinical signs observed in some animals were sedated behaviour and hunched posture, while ruffled fur and diarrhea was observed in all animals until day 10 and day 2 respectively. No obvious macroscopical organ findings were seen in any animal.

Conclusion

LD50 (males and female) > 2000 mg/kg bw