Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-22 to 2013-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

EC Number:

700-936-6

Molecular formula:

C17H11F2N7Na2O7S2

IUPAC Name:

tetrasodium 4-[(E)-2-[2-(carbamoylamino)-4-[(2,6-difluoropyrimidin-4-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate 4-[(E)-2-[2-(carbamoylamino)-4-[(4,6-difluoropyrimidin-2-yl)amino]phenyl]diazen-1-yl]benzene-1,3-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: 8 - 9 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 165 – 185 g (mean: 177.08 g, ± 20% = 141.67 – 212.50 g)
females: 140 – 160 g (mean: 148.25 g, ± 20% = 118.60 – 177.90 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF).
According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/-10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0902)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fiber bedding (lot no. 011012).
- Certificates of food, water and bedding are filed at BSL BIOSERVICE.
- Adequate acclimatisation period (at least five days)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage.
The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
In consultation with the sponsor the following doses (Table 1) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight
The animals were treated with the test item or vehicle on 7 days per week for a period of 14 days.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The animals were treated with the test item or vehicle on 7 days per week for a period of 14 days.

Frequency of treatment:

The animals were treated once daily.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 mg/kg bw, 300 mg/kg bw and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

24 animals (12 males and 12 females) were used for the study (3 male and 3 female animals per group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: random
- Section schedule rationale : alle male animals together and all female animals together

Examinations

Observations and examinations performed and frequency:

Body Weight and Food Consumption

The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the
treatment period as well as on the day of necropsy.
Food consumption was measured on study days 1, 8 and 14 for each animal.

Clinical Observations
All Animals were observed for clinical signs during the entire treatment period of 14 days.
General clinical observations were made at least once a day, approximately at the same time each day and considering the peak period of
anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity
and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia,
vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made
outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
 
Haematology
Haematological parameters (HCT, HB, RBC, PLT, WBC) were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in EDTA-coated tubes.

Sacrifice and pathology:

Pathology
On study day 15, all surviving animals of the study were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24139, expiry date: 06/2014
and xylazin, Serumwerk, lot no. 00512, expiry date: 07/2014) and were subjected to a detailed gross necropsy which included careful examination
of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight
No organ weights were taken in this study.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. Due to the small size of groups a statistical evaluation of the results was not performed in this study.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Mortality
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Observations
No signs of toxicity were observed in any animal during the whole observation period.

Body Weight Development
Throughout the treatment period, body weights were within the normal range of variation for this strain and no difference could be found
between C and treatment groups.

Food Consumption
In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress
of the study in all groups.
No considerable effect of Reactive Golden Yellow HF-RN 1331 on food consumption was found in any of the treatment groups of male female animals.

Haematology
No difference in haematological parameters was found for male animals at the end of the treatment period.
In female animals values of WBC were slightly decreased in MD (-22%) and HD (-33 %) groups. This could be test item related.
However, since no finding was found in male animals and since the standard deviation was relatively high (due to a low number of animals),
a clear statement cannot be given. A gender related difference is unlikely but cannot be excluded.
 
Pathology
No gross pathological changes were recorded for any animal of any group.

Effect levels

Basis for effect level:

other: see 'Remark'

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the repeated oral application of the test item Reactive Golden Yellow HF-RN 1331 to male
and female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight for 14 days was associated with no signs of toxicity or mortality.
Based on the data generated from this dose range finding study, dose levels of 100 300, and 1000 mg/kg body weight per day are suggested
for the subsequent main study with Reactive Golden Yellow HF-RN 1331.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Reactive Golden Yellow HF-RN 1331

via oral administration to rats over a period of 14 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 14 days.

Animals of an additional control group were handled identically as the dose groups but received aqua ad injectionem, the vehicle used in this study.

The 4 groups comprised 3 male and 3 femaleWistarrats.

During the period of administration, the animals were observed precisely each day for signs of toxicity.

Body weight and food consumption were measured weekly. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy.

The following doses were evaluated:

Control: 0 mg/kg body weight; Low Dose: 100 mg/kg body weight; Medium Dose: 300 mg/kg body weight; High Dose: 1000 mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad injectionem and administered daily during

a 14-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurement.

Summary Results

All animals survived until the end of the study without showing any signs of toxicity.

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

No signs of toxicity were observed in any animal during the whole observation period.

Throughout the treatment period, body weights were within the normal range of variation for this strain and no difference could be found between C and treatment groups.

No considerable effect ofReactive Golden Yellow HF-RN 1331on food consumption was found in any of the treatment groups of male female animals.

No difference in haematological parameters was found for male animals at the end of the treatment period.

In female animals values of WBC were slightly decreased in MD (-22%) and HD (-33 %) groups.

No gross pathological changes were recorded for any animal of any group.

 

Conclusion

Under the conditions of the present study, the repeated oral application of the test item Reactive Golden Yellow HF-RN 1331 tomale and female Wistarrats at doses of 100, 300 and 1000 mg/kg body weight for 14 days was associated with no signs of toxicity or mortality.

Based on the data generated from this dose range finding study, dose levels of 100 300, and 1000 mg/kg body weight per dayaresuggested for the subsequent main study withReactive Golden Yellow HF-RN 1331.