N'-(1,3-dimethylbutylidene)-3-hydroxy-2-naphthohydrazide

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:

november 2000 - december 2000

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:

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

Version / remarks:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): BMH
- Physical state/Description: white crystal
- Storage condition of test material: In the refrigerator in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 190-225 grams; females: 162-183 grams
- Fasting period before study: no
- Housing: group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55x34x21.5 cm). During activity monitoring animals were individually housed overnight in Macrolon plastic cagaes (type III, height 15 cm) with sterilised sawdust provided as bedding.
- Diet : free access to standard pelleted laboratory animal diet (from Altromin code VRF-1, Lage Germany)
- Water: free access to tap water
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 15 November 2000 To: 13 December 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing. Adjustment was made for specific gravity of the vehicle. Dose volume was 5 mL/kg bw. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of week 1 formulations were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 300689).

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment based on the results of a 5-day range finding study

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, and once prior to to start of treatment and on days 8, 15, 22 and 28 also performed outside the home cage in a standard arena. Symptoms were graded according to fixed scales(grade 1-4), with time of onset, degree and duration recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 8, 15, 22, 28

FOOD CONSUMPTION:
- Food consumption was determined weekly.

WATER CONSUMPTION: no quantitative investigation (only subjective appraisal), as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: all
- Parameters according to OECD guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters according to OECD guidelines were examined.

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to guideline
HISTOPATHOLOGY: Yes, according to guideline

Statistics:

The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-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 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of
significance. Group means were calculated for continuous data and medians were calculated for
discrete data (scores) in the summary tables. No statistical analysis was performed on motor activity data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no mortality occurred during the study period; salivation was observed in a dose-related fashion (commonly noted in rats of this age and strain following oral gavage, and not considered to be sign of systemic toxicity)

Mortality:

no mortality observed

Description (incidence):

no mortality occurred during the study period; salivation was observed in a dose-related fashion (commonly noted in rats of this age and strain following oral gavage, and not considered to be sign of systemic toxicity)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slighltly lower body weight gain in females in week 4

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no treatment related effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

high dose females showed slightly decreased prothrombine time and slightly increased RDW values

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

effects on ALAT, ASAT, bilirubin in males and females; effects on glucose and cholesterol in females

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

no changes observed in the FOB tests

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

in high dose group increased weights of liver (m/f) and kidneys and spleen (m)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hypertrophy in liver of high dose males and females; dose related increase in kidney cortical hyaline droplets in males

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: no mortality and no treatment-related clinical signs were observed. Observation of salivation is a common noted sign of rats of this age and strain following oral gavage. As the observation was not accompanied by microscopic abnormalities in the salivary gland, salivation is not considered to be toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN: High dose females showed a slightly decreased body weight gain in week 4

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

OPHTHALMOSCOPIC EXAMINATION: no toxicologically relevant effects observed in the FOB

HAEMATOLOGY: In high dose females a slight decrease in prothrombine time was observed (94% of control), and a slight increase in red cell distribution with (RDW) (107% of control).

CLINICAL CHEMISTRY: In mid and high dose animals, bilirubin levels were increased in a dose-dependent way (159-328% of control). In high dose males, a decreased level of ALAT (68%) and an increased level of ASAT (133%) was observed. In high dose females an increased level of ASAT (114%) and decreased level of plasma glucose (82%) was obsereved, whereas in mid-and high dose females decreased levels of ALAT (72 and 37%, respectively, dose-related) and increased levels of cholesterol were observed (143-149% of control).

NEUROBEHAVIOUR: no substance-related effects observed in the FOB

ORGAN WEIGHTS: In males of the high dose group, absolute and relative (to body weight) liver (113/110%) and kidney weights (125/121%) were increased. In high dose females relative liver weight (113% of control) was increased. Observed decreases in thymus weight in males and females were without dose-relationship, and in the absence of accompanying effects considered not toxicologically relevant.

GROSS PATHOLOGY: no treatment related observations.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver of high dose animals (3/sex) midzonal/centrilobular hypertrophy (minimal to slight) was observed. In the kidneys of males, there was a dose-related increase in incidence and severity in cortical hyaline droplets observed, which were minimal to slight in the low dose group (3 males), minimal to slight in the mid dose group (4 males) and moderate in all 5 high dose males. These droplets are considered to be alpha-2 globulin, a protein relevant for male rats. As this protein is not present in human, and in the absence of accompanying toxicological effects, increased incidence in hyaline droplets in male rats is not considered for risk assessment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: dose-related increase in plasma bilirubin levels in mid and high dose rats, and in females of the mid and high dose groups a dose-related decrease in plasma ALAT levels and an increase in plasma cholesterol levels.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Subacute 28-day oral toxicity with BMH was studied by daily gavage in rats with doses of 0, 50, 150 and 1000 mg/kg bw/day. Dose-related increase in plasma bilirubin levels in mid and high dose rats were observed. Females of the mid and high dose groups showed a dose-related decrease in plasma ALAT levels and an increase in plasma cholesterol levels. Observed cortical hyaline droplets in male kidneys were not accompanied by evidence of kidney damage. As cortical hyaline droplets are a common observation in male rats, and not observed in human kidney, this observation is not considered for human risk assessment.
Based on the observed effects on clinical biochemical parameters in mid and high dose rats, the NOAEL is set at 50 mg/kg bw/day.