Reaction mass of 6-(2-ethyl-6-methylanilino)-5-(4-{4-[ethyl(3-sulfobenzyl) amino] phenylazo} phenylazo)-4-hydroxynaphthalene-2-sulfonic acid, sodium and/or disodium salt(s) and 6-(2-ethyl-6-methylanilino)-5-(4-{4-[ethyl(4-sulfobenzyl) amino] phenylazo} phenylazo)-4-hydroxynaphthalene-2-sulfonic acid, sodium and/or disodium salt(s)

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acid Navy RN-2682

IUPAC Name:

Acid Navy RN-2682

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanRcc

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 250 mg/kg bw/day
Male: 5 animals at 1000 mg/kg bw/day
Female: 5 animals at 0 mg/kg bw/day
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 250 mg/kg bw/day
Female: 5 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimaitization and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.
At the end of the dosing period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.
From the animals of the low and middle dose groups, the liver (males only), lung, spleen, and the bone marrow (femur) were examined to establish a no-effect level.

Results and discussion

Results of examinations

Description (incidence and severity):

No test item-related signs of adverse toxicity were noted at any dose level during daily observations and no test item-related changes of adverse toxicity were noted during weekly behavioral observations (weeks 1-3).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

A trend toward lower production and increased turnover of red blood cells was noted in males treated with 1000 mg/kg/day, although nearly all values remained within the range of the historical control values.
Females at 1000 mg/kg/day and both sexes treated with 250 mg/kg/day or 50 mg/kg/day were unaffected.

Description (incidence and severity):

At 1000 mg/kg/day, slightly elevated lactate dehydrogenase levels were noted in males and females. The differences noted did not attain statistical significance and were considered to be the result of the mild increase in red blood cell turnover described above.

Description (incidence and severity):

Functional observational battery
No test item-related changes were noted during functional observation battery (week 4).

Grip strength
The mean fore- and hind limb grip strength values of the test item-treated rats compared favorably with those of the controls.

Locomotor activity
Minor differences between the mean locomotor activity of the test item-treated rats and control rats were considered to be within the range of typical background variation.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Minor differences in some mean absolute and relative organ weights were not supported by microscopical changes and were therefore considered to be incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of test item-unrelated background findings were noted. None of the observed findings were considered to be of toxicological relevance.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings considered to be test item-related were recorded in following organs:
• liver: minimal hepatocellular hypertrophy in two male animals treated with 1000 mg/kg/day. The hepatocellular hypertrophy was considered to be of adaptive character.
• lung: increased mean severity grade of alveolar macrophages in animals treated with 250 mg/kg/day and 1000 mg/kg/day, and increased incidence of alveolar macrophages in animals treated with 1000 mg/kg/day. Further minimal brownish pigment in animals treated with 1000 mg/kg/day. These findings were deemed to be due to accidental aspiration.
• spleen: increased mean severity grade of erythropoiesis in animals treated with 50, 250 and 1000 mg/kg/day. Additional increased mean severity grade of hemosiderin in male animals treated with 250 and 1000 mg/kg/day, and in female animals at all dose levels.
• bone marrow (femur): increased erythropoiesis in two animals treated with 1000 mg/kg/day.

Details on results:

Test item-related findings were generally restricted to the highest dose level of 1000 mg/kg/day, in which a mild reduction in red cell production and increased red cell turnover were noted, with a concomitant elevation in lactate dehydrogenase levels. Test item-related microscopical changes included minimal hepatocellular hypertrophy in the livers of two males treated with 1000 mg/kg/day (considered to be an adaptive change).
Increased erythropoiesis in the spleens of animals treated with 50, 250 and 1000 mg/kg/day, and the bone marrow of one male treated with 1000 mg/kg/day was considered to be test item-related and adaptive to the changes in red cell production described above. Hemosiderin deposition in the spleens of male animals treated with 250 and 1000 mg/kg/day, and in female animals at all dose levels was also considered to result from the described changes in red cell production.
Increased mean severity grade of alveolar macrophages in the lungs of animals treated with 250 mg/kg/day and 1000 mg/kg/day, and increased incidence of alveolar macrophages in animals treated with 1000 mg/kg/day. Further minimal brownish pigment was noted in animals treated with 1000 mg/kg/day. These findings were deemed to be due to accidental aspiration, rather than to systemic toxicity.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL = 1000 mg/kg bw/day