Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17. 9. – 2. 10.2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Name of test material (as cited in study report): Solvent Yellow 124
Substance type: organic
Physical state: liquid
Appearance: dark yellow viscous liquid

Composition of test material, percentage of components:
- Analytical purity: 90.0 % (w/w)
- Impurities (identity and concentrations):
4´-[2-((hydroxy)ethyl)ethylamino]azobenzene 3.0 % (w/w)
1,1-bis(N-ethyl[4-(phenylazo)phenyl]aminoethan-2-oxy)ethan 2.5 % (w/w)
- Unknown impurities 4.5 % (w/w)

Lot/batch No.: S2408
Expiration date of the lot/batch: Unlisted
Stability under test conditions: stable
Storage condition of test material: During the study the test substance was stored in glass bottle at laboratory temperature.

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 152-160 g
- Fasting period before study: 20 h
- Housing: animal room with monitoring conditions – 3 animals of one sex in one plastic breeding cage Velaz T4
- Diet (e.g. ad libitum): ST 1 BERGMAN – standard pelleted diet ad libitum
- Water (e.g. ad libitum): drinking tap water ad libitum
- Acclimation period: min 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3°C
- Relative humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark

STUDY TIME SCHEDULE
Animal supply: 12. 09. 2012
Experimental part of study: 17. 09. - 02. 10. 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
VEHICLE
- Lot/batch no. (if required): 5211201
- Purity:pharmaceutical quality

MAXIMUM DOSE VOLUME APPLIED: The single volume of administered suspension was 1ml/100 g of animal body weight.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: (according to EU Method B.1tris, Annex 1D)
START: 2000 mg/kg – 3 females (Step No.1): 2 females died and one female was humanely killed ► 300 mg/kg – 3 females (Step No. 2): no death ► 300 mg/kg – 3 females (Step No. 3): no death ► END of study
Doses:
2000 mg/kg b.w.
No. of animals per sex per dose:
3 females (step 1), 3 females (step 2), 3 females (step 3)
Control animals:
no
Details on study design:
Details on study design
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Body weight: before application, 8th day and before euthanasia of animals
Mortality: daily
Clinical examination:
the first day: twice (30 minutes and 3 hours after application)
the second day: twice (in the morning and in the afternoon) and daily thereafter for 14 days.

- Necropsy of survivors performed: yes

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
At the dose of 2000 mg/kg: 2 females died and one female was humanely killed by reason of moribund condition on the 3rd day morning
At the dose of 300 mg/kg: no death
Clinical signs:
2000 mg/kg:
The clinical signs of intoxication (piloerection, gibbous posture) were detected 30 minutes after the 1st day of application in all three animals. The skin, fur and visible mucous membranes coloured by test substance, gibbous posture, lurch tiptoe up gait, abdomen up and head down position, decreased response to stimuli, red discharge from nostril, tachypnoea, diarrhoea (excrements and bedding coloured by the test substance) were detected 3 hours after application.
The 2nd day of application in all three animals skin, fur and visible mucous membranes coloured by the test substance, gibbous posture, immobility, without reaction to stimuli, dyspnoea, diarrhoea (excrements and bedding coloured by the test substance) were diagnosed.

300 mg/kg: Clinical signs of intoxication (skin, fur and visible mucous membranes coloured by the test substance) were observed from 2nd to 5th day of application in all six animals (groups 2,3).
Body weight:
Body weight was recorded and weight increments were calculated in all surviving animals at the end of study. The test substance administered at the dose of 2000 mg/kg caused cachexy in all three animals.
The weight increments of animals at the dose 300 mg/kg were adequate to species, sex and age of animals in experiment.
Gross pathology:
2000 mg/kg:
Changes observed in all animals: skin, fur and visible mucous membranes coloured by the test substance; red discharge from nostril; cachexy; subcutaneous and skeletal muscle coloured by the test substance; dilatation and haemorrhagic inflammation (erosion) of stomach; chymus and intestines coloured by the test substance, blood in chymus; spleen decreased and pale; urine dark coloured.
300 mg/kg: The pathologic macroscopic changes (fat coloured by the test substance) were diagnosed during pathological examination

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the study results the value of LD50 of the test substance, Solvent Yellow 124, for rats is in the range > 300 mg/kg to ≤ 2000 mg/kg.
Executive summary:

The aim of the study was to investigate acute toxic effects of the test substance Solvent Yellow 124, after a single oral administration to Wistar rats.

The testing was performed according to the Method B.1 tris: Acute Oral Toxicity - Acute Toxic Class Method, Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

The test substance was administered in a single dose as solution in vehicle (olive oil), given orally via gavage to two groups of three female Wistar rats. The dosing was performed sequentially in three groups of three females: group No. 1 (first step) using the starting dose of 2000 mg/kg body weight, group No. 2 (second step) and group No. 3 (third step) using a dose of 300 mg/kg body weight. The volume of administered solution was 1 ml/100 g body weight of animals.

The test substance administered at the dose of 2000 mg/kg caused death of two animals and one animal was humanely killed by reason of moribund condition on the 3rd day morning.

The clinical signs of intoxication (piloerection, gibbous posture) were detected 30 minutes after the 1st day of application in all three animals. The skin, fur and visible mucous membranes coloured by test substance, gibbous posture, lurch tiptoe up gait, abdomen up and head down position, decreased response to stimuli, red discharge from nostril, tachypnoea, diarrhoea (excrements and bedding coloured by the test substance) were detected 3 hours after application. The 2nd day of application in all three animals skin, fur and visible mucous membranes coloured by the test substance, gibbous posture, immobility, without reaction to stimuli, dyspnoea, diarrhoea (excrements and bedding coloured by the test substance) were diagnosed. During pathological examination the following changes were observed in all animals: skin, fur and visible mucous membranes coloured by the test substance; red discharge from nostril; cachexy; subcutaneous and skeletal muscle coloured by the test substance; dilatation and haemorrhagic inflammation (erosion) of stomach; chymus and intestines coloured by the test substance, blood in chymus; spleen decreased and pale; urine dark coloured.

The test substance administered at the dose of 300 mg/kg caused no death in any of two administered groups of females (group No.2 and No. 3).

Clinical signs of intoxication (skin, fur and visible mucous membranes coloured by the test substance) were observed from 2nd to 5th day of application in all six animals.

The pathologic macroscopic changes (fat coloured by the test substance) were diagnosed during pathological examination. 

According to the study results the value of LD50 of the test substance, Solvent Yellow 124, for rats is in the range > 300 mg/kg to ≤ 2000 mg/kg.