Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Endpoint summary

Description of key information

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 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:

Experimental Starting Date - 18 August 2015; Experimental Completion Date - 08 September 2015; Study Completion Date - 03 November 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

Identification: FAT 21021/E TE
Batch: 72
Purity: ≥75 %
Physical state/ Appearance: Red solid
Expiry date: 16 February 2020
Storage Conditions: room temperature in the dark.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non- pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20 % of the mean body weight at the start of treatment.
The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study. The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 %, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Justification: Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Test Item Formulation and Experimental Preparation
For the purpose of the study the test item was freshly prepared, as required, as a suspension in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Procedure
Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose. A single animal was treated. In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated. A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.
Dose volume: 10 ml/kg.
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily. Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14. At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period. The faeces were stained black or dark red during the study.

Gross pathology:

No abnormalities were noted at necropsy.

Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
						Fº	Fº	Fº	Fº	Fº
2000	1-0 Female	0	0	0	0	0F	0F	0F	0F*	0F*	0F*	0F*	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-3 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

F =   Black stained faeces

F* = Dark red stained faeces

F° = Black/red stained faeces in cage

 

Individual Body Weights and Body Weight Changes

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
2000	1-0 Female	145	166	181	21	15
	2-0 Female	173	193	207	20	14
	2-1 Female	167	180	187	13	7
	2-2 Female	172	196	204	24	8
	2-3 Female	160	170	186	10	16

 

 Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Female	Killed Day 14	No abnormalities detected
	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Killed Day 14	No abnormalities detected
	2-2 Female	Killed Day 14	No abnormalities detected
	2-3 Female	Killed Day 14	No abnormalities detected

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight.

Executive summary:

A key study was conducted to assess the acute toxicity of the test substance FAT 21021/E according to OECD Guideline 420 and EU method B.1 bis (Acute Oral Toxicity – Fixed Dose Procedure). Following a sighting test at a dose level of 2000 mg/kg bw, additional four fasted female animals were given a single oral dose of test item, as a suspension in arachis oil BP, at a dose level of 2000 mg/kg bw. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

No mortality was observed at the end of the 14-day observation period. There were no signs of systemic toxicity. Black or dark red stained faeces were noted in all animals. All animals showed expected gains in body weight. No abnormalities were noted at necropsy.

In conclusion, based on the findings of the study, the acute median lethal oral toxicity (LD50) of FAT 21021/E to rats was found to be greater than 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Good quality study: Klimisch rating 1

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity – oral

An OECD test guideline GLP-compliant study was conducted to determine the acute oral toxicity FAT 21021. Following a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were given a single oral dose of test item, as a suspension in arachis oil BP. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths and no signs of systemic toxicity observed, except for black or dark red stained feces were noted in all animals. All animals showed expected gains in body weight. No abnormalities were noted at necropsy. The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight.

Acute toxicity – inhalation
Currently no study to assess the acute inhalation toxicity potential of Acid Red 260 is available. The melting point of Acid Red 260 was found to be greater than 350 °C indicating low vapour pressure. Hence, the substance is considered to have low volatility. Synthesis and spray drying of this chemical is performed in a closed process; the final product consists of non-dusty granules. Hence, the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur. Based on column 2, ‘Specific rules for adaptation from column 1’ of the table given in REACH Annex VII, the study on acute inhalation toxicity only needs to be conducted if an exposure via inhalation is to be expected, based on vapour pressure and/or the likelihood of an exposure to aerosols, particles or droplets. Referring to the expected low volatility of the substance, the fact that the chemical is imported into the EU in a formulated form as a dust-free powder or as a granulate, the exposure via inhalation is considered to be unlikely. Further, the chemical is found to have water solubility of 1.2 g/L, hence, in the case of dust of the substance entering the respiratory tract, it will be trapped in the mucus and cleared, thereby further limiting the absorption. The chemical showed low toxicity potential in the available acute oral toxicity study (LD50: > 2000 mg/kg bw), hence, it does not need to be classified STOT SE and low toxicity is expected for this chemical via the inhalation route. Taking above arguments into account, low toxicity potential is expected on acute exposure of Acid Red 260 via inhalation route and hence testing by the inhalation route was considered scientifically not necessary.

 

Acute toxicity: dermal

Currently no study to assess the acute dermal toxicity potential of Acid Red 260 is available. The molecular weight of Acid Red 260 is 1083.06 g/mol, indicating it being too large for dermal absorption. It has water solubility of 1.2 g/L and n-octanol/water partition coefficient (log P) of -2.02, indicating it being too hydrophilic to cross the lipid rich environment of the stratum corneum. Hence, the dermal uptake for the substance will be low. The chemical showed low toxicity potential in the available acute oral toxicity study (LD50: > 2000 mg/kg bw), hence, it does not need to be classified as STOT SE. Similarly, absence of systemic toxicity in skin irritation and sensitization studies, further supports the conclusion that no adverse effects are expected for the chemical via the dermal route. Further, experience with similar chemical substances has demonstrated that it is very unlikely that toxicity related to the intrinsic properties of the chemical only show up upon dermal exposure and not after systemic application. Taking above arguments into consideration, low toxicity is expected on acute dermal exposure of Acid Red 260 and testing by the dermal route was considered scientifically not necessary.

Justification for classification or non-classification

Based on the observed LD50 of >2000 mg/kg bw in the acute oral toxicity study, Acid Red 260 does not need to be classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.