Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Version / remarks:
2001
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate
EC Number:
272-559-0
EC Name:
Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate
Cas Number:
68877-33-8
Molecular formula:
C28H21N9O7S2.2Na
IUPAC Name:
Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5- hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks of age
- Weight at study initiation:
- Fasting period before study: overnight
- Housing: In groups of up to four in suspended solid-floor polypropylene cages furnished with softwood flake bedding.
- Diet: free access to 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water: free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): not specified
- Justification for choice of vehicle: solubility of the test item

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
Doses:
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose and a single animal was treated.
In the absence of toxicity at a dose level of 300 mg/kg, an additional animal was treated at a dose level of 2000 mg/kg.
In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of 4 animals was treated at a dose level of 2000 mg/kg.
No. of animals per sex per dose:
1 female, 300 mg/kg
5 female, 2000 mg/kg
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for up to 14 days. Morbidity and mortality checks were made twice daily, early and late during normal working days, and once daily at weekends and public holidays. Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: 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.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
act. ingr.
Mortality:
Dose Level - 300 mg/kg: there were no deaths
Dose Level - 2000 mg/kg : There were no deaths.
Clinical signs:
Dose Level - 300 mg/kg: No signs of systemic toxicity were noted during the observation period
Dose Level - 2000 mg/kg : No signs of systemic toxicity were noted during the observation period. Black stained feces were noted in one treated animal one to four days after dosing. The animal appeared normal five days after dosing.
Body weight:
Dose Level - 300 mg/kg: The animal showed expected gains in body weight over the observation period.
Dose Level - 2000 mg/kg : All animals showed expected gains in body weight over the observation period.
Gross pathology:
Dose Level - 300 mg/kg: No abnormalities were noted at necropsy.
Dose Level - 2000 mg/kg : Patchy pallor of the liver was noted in one treated animal. No abnormalities were noted at the necropsy of the four other animals.

Any other information on results incl. tables

Appendix 1 Individual Clinical Observations and Mortality Data - 300 mg/kg






















































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



300



1-0


female



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



Appendix 2 Individual Body Weights and Body Weight Changes - 300 mg/kg




























Dose level (mg/kg)



Animal number and sex



Body Weight (g) at Day



Body Weight Gain (g)


During Week



0



7



14



1



2



300



1-0 female



208



212



221



4



9



Appendix 3 Individual Necropsy Findings - 300 mg/kg


















Dose Level (mg/kg)



Animal Number and Sex



Time of Death  



Macroscopic Observations


 



300



1-0 female



Killed Day 14



No abnormalities detected



Appendix 4 Individual Clinical Observations and Mortality Data - 2000 mg/kg










































































































































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



2000



2-0


female



0



0



0



0



F0



F0



F0



F0



0



0



0



0



0



0



0



0



0



0



3-0


female



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



3-1


female



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



3-2


female



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



0



3-3


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 = Feces stained black


Appendix 5 Individual Body Weights and Body Weight Changes - 2000 mg/kg




























































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



2-0


female



221



221



234



0



13



3-0


female



209



220



233



11



13



3-1


female



203



219



229



16



10



3-2


female



202



228



238



26



10



3-3


female



208



219



227



11



8



 


Appendix 6 Individual Necropsy Findings - 2000 mg/kg






































Dose Level (mg/kg)



Animal Number and Sex



Time of Death



Macroscopic Observations


 



2000



2-0


female



Killed Day 14



Liver: Patchy pallor



3-0


female



Killed Day 14



No abnormalities detected



3-1


female



Killed Day 14



No abnormalities detected



3-2


female



Killed Day 14



No abnormalities detected



3-3


female



Killed Day 14



No abnormalities detected


Applicant's summary and conclusion

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:

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat. Following a sighting test at dose levels of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a solution in distilled water, at a dose level of 2000 mg/kg body weight.  Clinical signs and body weight development were monitored during the study.  All animals were subjected to gross necropsy.

There were no deaths. There were no signs of systemic toxicity. All animals showed expected gains in body weight. Patchy pallor of the liver was noted at necropsy of one animal treated at a dose level of 2000 mg/kg.  No other abnormalities were noted.

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 (Globally Harmonized Classification System − Unclassified).