disodium 4-hydroxy-3-[2-[4-({4-[2-(1-hydroxy-4-sulfonatonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-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 2013-07-02

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:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

EC Number:

700-924-0

Molecular formula:

C41H30N4Na2O10S2

IUPAC Name:

disodium 4-hydroxy-3-[(E)-2-[4-({4-[(E)-2-(1-hydroxy-4-sulfonaphthalen-2-yl)diazen-1-yl]-3-methoxyphenyl}(phenyl)methyl)-2-methoxyphenyl]diazen-1-yl]naphthalene-1-sulfonate

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: males: 170 – 188 g (mean: 185 g, ± 20% = 148 – 222 g)
females: 125 – 154 g (mean: 137 g, ± 20% = 110 – 164 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:

cotton seed oil

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. 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.

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 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.

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
Discoloured red faeces were observed transiently from study day 8, during the second treatment week in 3/6 HD animals and 1/6 MD animals.
This was not associated with any clinical, haematological or macroscopic alterations and is assumed to be caused by the colour of the test item.
Red nasal discharge observed on 2 days after administration in one male animal of the MD group is not considered to be related to the test item.

Body Weight Development
In both males and female animals, the mean body weight increased with the progress of the study and remained within the normal range of variation for this strain. No considerable difference in body weight gain was observed when dose groups were compared to the control group.

Food Consumption
Acid Red RN 2949 had no effect on food consumption in this study. Throughout the treatment period no considerable difference in food
consumption was observed in male and female animals when dose groups were compared to the control group.

Haematology
Acid Red RN 2949 had no effect on haematological parameters determined at the end of the treatment period. Mean red cell parameters,
platelet and white blood cell counts were in the normal range of variation for male and female Wistar rats of this age.
There were no considerable differences between the dose groups and the control group.
A low WBC count observed in male animal no. 24 was not associated with any sign of pathology.

Pathology
At necropsy no macroscopic findings were found for any of the animals.

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, a repeated oral toxicity study with Acid Red RN 2949 administered to male and female Wistar rats
at doses of 100, 300 and 1000 mg/kg/day for 14 days, the following conclusions can be made.
The used dose levels of Acid Red RN 2949 were associated with no signs of toxicity. Thus, these dose levels are proposed for a subsequent
28-day repeated dose oral (gavage) toxicity study.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Acid Red RN 2949 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 cottonseed oil, 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

The test item formulation was prepared freshly on each day of administration. The test item was suspended in cottonseed oil 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

No mortality occurred in the control or any of the dose groups during the treatment period of this study. There were no considerable clinical symptoms and body

weight developed normally in all animals treated with Acid Red RN 2949. Food consumption was unaffected and no macroscopic alterations were found at

necropsy of the dosed animals. Red cell parameters, platelet and white blood cell counts were in the normal range at the end of the treatment period.

Conclusion

Under the conditions of the present study, a repeated oral toxicity study with Acid Red RN 2949 administered tomale and female Wistarrats at doses of

100, 300 and 1000 mg/kg/day for 14 days, the following conclusions can be made.

The used dose levels of Acid Red RN 2949 were associated with no signs of toxicity. Thus, these dose levels are proposed for a subsequent 28-day repeated dose oral (gavage) toxicity study.