Reaction mass of isomers disodium 8-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-5-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, disodium 5-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-8-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

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:

2013-03-07 to 2013-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Remarks:

Conducted on the basis of GLP in a certified and accredited GLP laboratory. Not audited by Quality Assurance unit and therefore the study does not have the GLP status.

Limit test:

no

Test material

Test material information

1

Reference substance name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

EC Number:

700-946-0

Molecular formula:

C40H35N7Na2O6S2

IUPAC Name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid: particulate/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: 156 – 182 g (mean: 169 g, ± 20% = 135.20 – 202.80 g)
females: 118 – 146 g (mean: 130.58 g, ± 20% = 104.47 – 156.70 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 were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose)
and 1 control group (C). The animals were treated with the test item or vehicle on 7 days per week for a period of 14 days.

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 in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

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, Pharma Novo, 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. All macroscopic findings were recorded and organs showing gross abnormalities were preserved in neutral buffered formalin.

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:
There were no mortality occurred in the treated and control group during the study.

Clinical Observations:
There were no clinical signs observed during the study in the treated or control groups except for slight salivation (reddish) in one animal (animal no. 4) of LD group. However, this was observed on one treatment day (day 3). No toxicological relevance was considered for this finding as this was observed in one isolated male of LD group on one particular day.

Body Weight Development:
In both males and females, there were no treatment related changes noted for body weight and body change during the study period.
However, there was slight increase in body weight change in male and female HD group on 1st week of treatment. There was slight decrease in body weight change noted in female HD group during 2nd week of treatment. The above changes did not show a dose response pattern and in addition the decrease in female HD group during 2nd week of treatment was due to considerable decrease in weight gain of one isolated female (no. 24). Therefore, the changes were not considered to have toxicological relevance.

Food Consumption:
In both males and females, there were no treatment related changes noted for food consumption during the study period. However, there was slight increase in mean food consumption of males in HD group. But the individual values appeared to be within the normal range of variations.
Hence, the changes were not likely to be related to the treatment.

Haematology:
In males and females, there were no test item related changes noted for RBC, Hb, HCT, PLT and WBC values.
However, there were changes noted for PLT, Hb and WBC values in male or female treated groups,
- There was increase in mean PLT value in Male HD group without the dose response pattern.
- There was increase in mean Hb value in female HD group assumed to be due to substantial increase in the value of a single isolated animal.
- There was increase in mean WBC values of male and female animals in dose related manner in LD, MD and HD groups with the individual values showing high heterogeneity.
The individual and mean values of PLT, Hb and WBC values were within historical control range.
Hence, the above changes were not likely to be related to the treatment.

Pathology:
At necropsy, there were no macroscopic changes noted in animals of treated and control groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

not determinable

Remarks:

no NOAEL identified

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 to male and female Wistar rats at doses of 100, 300 and 1000 mg/kg body weight for 14 days was associated with no mortality and no clear sign of toxicity.
Based on the data generated from this dose range finding study, dose levels of 100, 300 and 1000 mg/kg body weight per day could be considered for the subsequent main study with the test item.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of test item 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 receivedaqua 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. At the conclusion of the test, all animals were sacrificed  and observed macroscopically.
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
Hematological parameters (RBC, WBC, HCT, Hb and PLT) were measured in all animals of control and treated groups at the end of the treatment.
The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionemand administered daily during a 14-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measuremen

Summary Results
No mortality occurred in the control or any of the dose groups during the treatment period of this study.
No clear test item related clinical symptom with toxicological relevance was observed.
No influence on body weight, body weight gain and food consumption was observed.
No test item related influence on parameters of haematology was observed.
No specific gross pathological changes were recorded for the male and female animals at necropsy.

Conclusion
Under the conditions of the present study, the repeated oral application of the test item to male and female Wistarrats at doses  of 100, 300 and 1000 mg/kg body weight for 14 days was associated with no mortality and no clear sign of toxicity.
Based on the data generated from this dose range finding study, dose levels of 100, 300 and 1000 mg/kg body weight per day could be considered for the subsequent main study with the test item.