Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 January 2019 to 21 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%

Test animals

Species:
rat
Strain:
other: RccHan: WIST albino
Sex:
female
Details on test animals and environmental conditions:
Test Item Preparation and Analysis
3.2.1 Formulation
The test item was formulated at concentrations of 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg body weight. The test item formulations were prepared on the day of dosing. The absorption of the test item was not determined. Determination of the homogeneity, stability and purity of the test item or test item formulations were not undertaken as part of this study. Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

3.3 Animal Information
Healthy nulliparous and non-pregnant female RccHan™:WIST albino rats were obtained from Envigo RMS (UK) Ltd. The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of one or four rats of the same sex. Each animal was identified uniquely within the study by tail marking. Each cage label was color-coded and was identified uniquely with the study number, dose level and animal mark. The animals were allowed to acclimatize to the conditions described below for at least seven days before treatment. For those animals selected for this study, their body weights were in the range 161 to 181 g and they were approximately eight to twelve weeks of age prior to dosing (Day 1). The body weight variation did not exceed ± 20% of the mean body weight of any previously treated animals. 3.4 Animal Care and Husbandry Animals were housed inside a limited access rodent facility (Building F21, Room 047). The facility was designed and operated to minimize the entry of external biological and chemical agents and to minimize the transference of such agents between rooms. The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 20 to 24C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study.
Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data. Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to beautomatically brought into operation should the public supply fail.
The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fiber bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals. The animals were allowed free access to a standard rodent diet (Teklad 2014C Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
Each cage of animals was provided with an Aspen chew blocks or balls for environmental enrichment. Chew blocks or balls were provided throughout the study and were replaced when necessary.
Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
Each batch of diet was analyzed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinized and approved before any batch of diet was released for use.
The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the chew blocks. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analyzed, as none that may have interfered with or prejudiced the outcome of the study was known.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Dose Administration
The appropriate dose volume of the test item was administered to each rat by oral gavage using a plastic syringe and plastic catheter.
A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.
Formulations were stirred before and throughout the dosing procedure.
Doses:
300 mg/kg
2000 mg/kg
No. of animals per sex per dose:
6 female rats
Control animals:
no
Details on study design:
Study Design
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the
starting dose.
Two single animals were treated as follows:

Dose level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Number of rats
Female
300 30 10 1
2000 200 10 1


In the absence of mortality or toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated as follows:

Dose level (mg/kg) Concentration (mg/mL) Dose volume (mL/kg) Number of rats
Female
2000 200 10 4

A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

Results and discussion

Preliminary study:
In the sighting study, red faeces was observed on Day 2 for the female dosed at 300 mg/kg and on Day 6 for the female dosed at 2000 mg/kg. Recovery, as judged by external appearance and behaviour, was complete by either Day 3 or Day 7.
Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other:
Remarks:
The acute median lethal oral dose greater than 2000 mg/kg
Mortality:
There were no deaths during the study.
Clinical signs:
In the sighting study, red faeces was observed on Day 2 for the female dosed at 300 mg/kg and on Day 6 for the female dosed at 2000 mg/kg. Recovery, as judged by external appearance and behaviour, was complete by either Day 3 or Day 7.

In the main study at 2000 mg/kg, red faeces was observed on Day 2 for one female and on Day 3 for all four females. Also, on Day 4 orange faeces was observed for all four animals. Recovery, as judged by external appearance and behaviour, was complete by Day 5.
Body weight:
All animals were considered to have achieved satisfactory body weight gains throughout the study.
Gross pathology:
Macroscopic examination at study termination on Day 15 revealed pallor of liver in one female (animal number 401) dosed at 300 mg/kg in the sighting study and abnormal content in the stomach (dark, hard and approximately 3 x 3 mm) in one animal (animal 404) dosed at 2000 mg/kg in the main study. No abnormalities were revealed in any other animal at the macroscopic examination at this time.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute median lethal oral dose (LD50) to rats of Acid Orange 94 Refined was demonstrated to be greater than 2000 mg/kg body weight.
Executive summary:

The acute median lethal oral dose (LD50) to rats of Acid Orange 94 Refined was determined according to the

OECD Guideline for Testing of Chemicals No.420 ‘Acute Oral Toxicity – Fixed and EEC Methods for the determination of toxicity and other health effects. Commission Regulation No. 440/2008, Part B, Method B.1 bis. Acute Oral Toxicity: Fixed Dose Procedure. 30 May 2008. Dose Method’ Adopted 17 December 2001, according to GLP.

‘The melting point was determined using differential scanning calorimetry according to the OECD 102 guideline in accordance with GLP. The test item was determined to decompose from approximately 176 °C (449 K). As the test item decomposed, no value for melting point could be determined.’

The fixed dose method is a stepwise procedure that uses sighting investigations of one animal of a single sex per step followed by a main study of a further four animals. Depending on the mortality and/or the moribund status of the animals, on average two sighting steps and one or two main study steps may be necessary to allow a judgment on the acute toxicity of the test item.

The acute median lethal oral dose (LD50) to rats of Acid Orange 94 Refined was demonstrated to be greater than 2000 mg/kg body weight.

According to the Globally Harmonised System (GHS), described in Annex 2, Acid Orange 94 Refined does not meet the criteria for classification.