Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

EC Number:

226-103-2

EC Name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Cas Number:

5280-68-2

Molecular formula:

C33H27ClN4O6

IUPAC Name:

N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-{[2-methoxy-5-(phenylcarbamoyl)phenyl]diazenyl}-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): Repeated dose 28-day oral toxicity study by daily gavage in the rat followed by a 14-day recovery period: Permanent Carmin FB B02
Analytical purity: 96.26%

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Crl:WI(Han) (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight range in groups at day 1: males: 170 g - 200 g, females: 135 g - 150 g
- Housing: in groups of 5 in macrolon cages (MIV type, during overnight activity monitoring individual housing in MIII type)
- Diet: Rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest , Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- dose formulations were prepared daily within 4 h prior to dosing and were homogenized to a visually acceptable level.
- the test item is stable in dose formulations for up to 96 h.

VEHICLE
- water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase on samples as specified below, using a method that has been validated under NOTOX project 488240.
group I: accuracy (middle position of container)
group II: accuracy and homogenicity (top, middle, botton position of container)
group III: accuracy (middle position of container)
group IV: accuracy and homogenicity (top, middle, botton position of container)

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

- 5 male and 5 female in each dose group
- 5 male and 5 female for recovery groups at dose 0 and 1000 mg/ kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 5-days dose range finding study with the test item
- Post-exposure recovery period in satellite groups: the duration of recovery was 14 days

Positive control:

- none

Examinations

Observations and examinations performed and frequency:

MORTALITY/VIABILITY: Yes
- twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to treatment and at weekly intervals, this was performed outside the home cage in a standard arena.

BODY WEIGHT: Yes, weekly

FOOD CONSUMPTION: yes, weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes
- Parameters checked see below in Hematology
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks (the latter only applied for recovery groups)
- Animals fasted: Yes (with a maximum of 20 h before blood collection)
- How many animals: all animals
- Parameters checked see below

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks and after 6 weeks (the latter only applied for recovery groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked see below

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (parameters checked see below)
HISTOPATHOLOGY: Yes (parameters checked see below)

Statistics:

- If the variables can be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel test was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied fo frequency data.

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:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
- no deaths occurred during the test
- red feces were noted in animals treated with the test item and/or between days 1 and 3 of the recovery period.
- one animal showed red staining of the back. probably caused by staining by the test substance.
- overall no clinical observations of toxicological relevance were found

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weight and body weight gain were noted.
CLINICAL LABORATORY INVESTIGATOIONS
- HAEMATOLOGY
- No changes of toxicological relevance were observed in the hematology parameters after the treatment or recovery periods in both males and females.
- Some changes in haematological parameters were noted in individual animals that distinguished treated animals from control animals. These changes were considered to be of no toxicological significance as they occured in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain and/or occurred only at the end of the recovery phase.

- CLINICAL CHEMISTRY
Several clinical biochemistry parameters were changed at a dose level of 1000 mg/kg/day. These changes were slight and within the range considered to be normal. Therefore these changes were not considered to be of toxicological relevance.
No changes of toxicological relevance were noted in the clinical biochemistry parameters after the treatment and the recovery periods.

- URINALYSIS
No changes of toxicological relevance were observed in the urinalysis parameters after the treatment with the test item and the recovery period in both males and females, at any dose level tested.
Some changes were noted in individual animals that distinguished treated animals from control animals. These changes were considered to be of no toxicological significance since no higher potassium level was noted at the end of the treatment period and no corresponding microscopic findings were found, this was considered of no toxicological relevance.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios.
Some changes were noted in individual animals that distinguished treated animals from control animals. These changes were within the range considered normal for rats of this age and strain. Therefore the results were considered to be of no toxicological significance.

GROSS PATHOLOGY
- reddish content of several parts of the intestinal tract was noted at the end of treatment in all animals at 250 and 1000 mg/kg/day. This was considered to be caused by staining by the test substance and therefore considered to be of no toxicological relevance.
- At the end of recovery greenish soft nodules in the epididymides were noted in two males at the highest concentration. This is a common finding in this strain and is not considered related to treatment.

HISTORICAL CONTROL DATA (if applicable)
- all other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no clinical signs; no mortality; body weight development was not impaired; food consumption was not affected; no findings in haematology; urinalysis, gross pathology and histopathology up to highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day was established for male and female rats.

Executive summary:

A 28 -day repeated dose toxicity study with the test item administered by gavage to Wistar rats (SPF-bred; 5/sex/dose) was performed according to OECD TG 407. The dose levels for this study were 50, 250 and 1000 mg/kg bw/day. A control group was treated similarly with the vehicle, water, only. The dosing lasted for 28 days and a 14 day recovery group for controls and the high dosage group (1000 mg/kg bw/d) was included. The test item revealed no treatment-related findings. From the results presented in this report a No Observed Adverse Effect Level(NOAEL) for the test item of 1000 mg/kg bw/day was established.