Trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

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:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: recent guideline study according to GLP, screening study for reprductive effects

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acid Brown 425

IUPAC Name:

Acid Brown 425

Constituent 2

Reference substance name:

Trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

EC Number:

278-145-6

EC Name:

Trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

Cas Number:

75234-41-2

Molecular formula:

C40H26CrN8O14S2.3Na

IUPAC Name:

trisodium bis[2-[[2,4-dihydroxy-3-[(2-methyl-4-sulphophenyl)azo]phenyl]azo]benzoato(3-)]chromate(3-)

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:

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 295 to 329 g (males) and 178 to 213 g (females)
- Identification: Cage card and individual animal number (ear tattoo).Pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values for both temperature and humidity were outside these ranges but were transient variations and were considered not to have any influence on the study and, therefore, these data are not reported but retained at Harlan Laboratories. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) (batch/lot nos. 02105120601 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Acid Brown 425 was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were divided into daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored in refrigerator (5 ± 3 °C) in brown bottles in daily aliquots.

Based upon the results of stability analyses performed within the Harlan Laboratories non-GLP study D51147 (Dose Range-Finding Study for a Reproduction/Developmental Toxicity Screen¬ing Test in the Han Wistar Rat), dose formulations were stable for at least one week in refrigerator.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg bw/day (group 1, control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D51147, using dose levels of 0, 100, 300 and 1000 mg/kg/ day. No adverse toxic effects were found at any dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (group 1), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (4 hrs at room temperature and 8 days in refrigerator). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the responsible person for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS following an analytical procedure developed at Harlan Laboratories study D51160. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

RESULTS

The application formulations investigated during the study were found to comprise Acid Brown 425 in the range of 96.4% to 113.1% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of Acid Brown 425 in the preparations was approved because single results found did not deviate more than 1.1% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours at room temperature or eight days refrigerated due to recoveries which met the variation limit of ±10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item Acid Brown 425 and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Minimum 4 weeks (males) or approximately 7 weeks (females)

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

VIABILITY/MORTALITY
Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

TERMINATION AND NECROPSY
Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

Histological examination of ovaries was carried out on any females that did not give birth.
A histopathology peer review was performed and the results included in the histopathology phase report.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals survived the scheduled study period. Black colored feces were recorded in females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item. In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in females at any dose level.

Mortality:

mortality observed, treatment-related

Description (incidence):

All animals survived the scheduled study period. Black colored feces were recorded in females of all groups treated with the test item from the start of dosing until necropsy. Severity of mid and high dose groups were increased. This finding was considered to be related to the treatment with the test item. In one female (no. 51) in the control group chromodacryorrhea of the left eye was recorded in the first four day of the pre-pairing period. No other clinical signs were noted in females at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Discoloration of ovaries was recorded for two females in the 100 mg/kg body weight/day dose group and for one female in the 300 mg/kg body weight/day dose group. This finding was considered to be incidental

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings recorded were considered to be within the range of normal background alterations.

Histopathological findings: neoplastic:

not examined

Details on results:

There were no test item-related effects found in any females in any dose group.
Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with marginal further decrease between mid and high dose groups despite the triplication of the dose.
There is indication of adaptive reactions with drecrease of effect over time.
The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

2

Basis for effect level:

other: No adverse effects at all

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:

There were no test item-related effects found in any females in any dose group.
Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with marginal further decrease between mid and high dose groups despite the triplication of the dose. There is indication of adaptive reactions with drecrease of effect over time. The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the evaluation of effects on food consumption, body weight and/or body weight gain in males the NOAEL for systemic toxicity for males was considered to be 1000 mg/kg body weight/day and the NOEL is 1000 mg/kg body weight/day for females.
Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item Acid Brown 425 to rats. The test material was administered in highly purified water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test material was administered to male rats for 28 days (except for one male up to 34 days) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (between 40 and 53 days for non-pregnant females and between 41 and 54 for dams).

Black colored feces seen in all test item-treated animals were considered to be a result of the staining properties of the dark brown test item.

There were no test item-related effects found in any females in any dose group.

Treatment with the test item at the high-dose level caused slight reductions in food consumption, body weights and body weight gains in males observed during the pre-pairing. The main difference being between low and mid dose groups with only marginal further decrease between mid and high dose groups despite the trippling of the dose. There is indication of adaptive reactions with drecrease of effect over time. The effects are considered test material related but not adverse, which is also suported by the absence of any effects in females even after prolonged exposure.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

No test item-related findings were noted in pups up to day 4 post partum at any dose level.

Based on the evaluation of effects on food consumption, body weight and/or body weight gain in males the NOAEL for systemic toxicity for males was considered to be 1000 mg/kg body weight/day and the NOEL is 1000 mg/kg body weight/day for females.

Based on the absence of effects on fertility rate, mating performance, number of pups born and post natal loss of pups up to day 4 post partum the NOEL for reproduction toxicity was considered to be 1000 mg/kg body weight/day.