.beta.-Alanine, N-[2-chloro-4-[2-(4-nitrophenyl)diazenyl]phenyl]-, 2-[2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)ethoxy]-2-oxoethyl ester

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:

From February 6 to April 3, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Yellow 981

IUPAC Name:

Yellow 981

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean measured concentrations of homogeneity samples were 99.8 %, 94.9 % and 93.6 % of the nominal concentrations of homogeneity samples (10, 40 and 200 mg/ml, respectively). Individual concentrations were within -2 % to +1 % of mean concentrations. The test item is therefore homogeneousy distributed in the vehicle.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

28 days of administration

Doses / concentrations 2

Dose / conc.:

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

Remarks:

28 days of administration + 14-day recovery period

Doses / concentrations 3

Dose / conc.:

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

Remarks:

28 days of administration

Doses / concentrations 4

Dose / conc.:

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

Remarks:

28 days of administration

Doses / concentrations 5

Dose / conc.:

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

Remarks:

28 days of administration

Doses / concentrations 6

Dose / conc.:

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

Remarks:

28 days of administration + 14-day recovery period

No. of animals per sex per dose:

5 male and 5 female animals per group

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Mortality (daily), clinical signs (daily), functional observation battery (during week 4), food consumption (weekly), body weight (weekly), haematology (immediately prior to sacrifice), biochemistry (immediately prior to sacrifice), urinalysis (18-hour fasting period immediately prior to sacrifice)

Sacrifice and pathology:

Organ weights, macroscopic and microscopic findings

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Bright orange faeces (day 2 onwards; 200 and 1000 mg/kg bw/day groups only; first two days of recovery period only)
- Slight salivation (1 female administered 1000 mg/kg bw/day, day 24 only; isolated finding)
- Miosis (1 control male during pretest only; considered temporary)

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Signs of anaemia: reduced mean RBC and Hb, increased mean cell volume and cell Hb (males and females administered 1000 mg/kg bw/day); decreased mean cell [Hb] (females adminsitered 1000 mg/kg bw/day)
- Signs of compensatory reticulocytosis: increased absolute and mean reticulocyte count (males and females administered 200 and 1000 mg/kg bw/day); left shift toward high fluorescence reticulocytes (males and females administered 200 and 1000 mg/kg bw/day); increased normoblast count (females administered 200 and 1000 mg/kg bw/day; statistically significant at 1000 mg/kg bw/day only)
- Signs of compensatory reaction to anaemia: abnormal RBC morphology (polychromatophilia) (males and females administered 1000 mg/kg bw/day and females administered 200 mg/kg bw/day)
- Recovery group: unaffected by test item

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Elevated total bilirubin (males and females administered 1000 mg/kg bw/day, and females administered 200 mg/kg bw/day)
- Reduced globulin (males and females administered 1000 mg/kg bw/day) and concomitant increased albumin:globulin ratio
- Increased phosphorus and sodium (females administered 1000 mg/kg bw/day)
- Lower urea (treated males; incidental; high control value)
- Elevated urea (females administered 200 mg/kg bw/day; not dose-dependent; incidental)
- Recovery group: elevated total bilirubin persisted (females only)

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Decreased mean specific gravity (males administered 1000 mg/kg bw/day; incidental and reversible in recovery period)

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Salivation (1 control male + 1 female administered 50 mg/kg bw/day; incidental)
- Grip strength significantly increased (males administered 200 and 1000 mg/kg bw/day; forelimbs only; incidental)
- Locomotor activity significantly decreased (females administered 1000 mg/kg bw/day; not test item related); other locomotor activity differences were considered not dose-related and incidental

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Increased relative and absolute spleen weights (males and females administered 1000 mg/kg bw/day; also in those administered 200 mg/kg bw/day but not statistically significant)
- Increased liver:bw ratio (females administered 50 and 1000 mg/kg bw/day only; incidental among those administered 50 mg/kg bw/day only)
- Recovery group: spleen and liver weight differences reversed (males and females); decreased absolute and relative thymus and ovary weights (females administered 1000 mg/kg bw/day; incidental)

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Minimal extramedullary haematopoiesis of liver (1 female administered 200 mg/kg bw/day and 1 male administered 1000 mg/kg bw/day)
- Slightly increased erythropoiesis of speen (males and females administered 1000 mg/kg bw/day and 3 males and 2 females administered 200 mg/kg bw/day)
- Recovery group: no significant findings

Details on results:

Oral administration to rats at doses of 50, 200 and 1000 mg/kg/day for 28 days resulted in no effects upon mortality, daily clinical observations, weekly behiavioural observations, functional observational battery, food consumption, body weight or urinalysis parameters.
Treatment-related findings were generally restricted to rats treated with 200 or 1000 mg/kg/day.
Changes were seen in hematology parameters (reduced mean red blood cell count, reduced mean hemoglobin levels, increased mean cell volume and increased mean cell hemoglobin in both sexes and reduced mean cell hemoglobin concentration in females, increased reticulocytes counts with 'left-shift', increased normoblast counts and polychromatophilia) and clinical biochemistry parameters (increased total bilirubin in both sexes, reduced globulin levels, increased albumin/globulin ratios, increased phosphorus and sodium in females), which were generally indicative of anemia with compensatory reticulocytosis and bone marrow stimulus.
These changes were reflected also in the results of histopathology, whereby increased erythropoiesis was noted in the spleen of one female treated at 200 mg/kg/day, and extramedullary hepatic erythropoiesis (generally associated with splenic findings) in the livers of one female treated at 200 mg/kg/day and in one male and three females treated with 1000 mg/kg/day.
No test item-related histopathologic findings were noted in rats following the 14-day recovery period.
After the recovery period, hematology parameters in males of males treated at 1000 mg/kg(day were considered to be unaffected by test item; the elevated lymphocyte count and prolonged activated partial thromboplastin time were considered to be incidental.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (sub-acute, rat, oral) = 50 mg/kg bw/day