2-butyl-1,3-diazaspiro[4.4]non-1-en-4-one hydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th December 1996 - 6 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: done under GLP and OECD method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline No. 407 "Repeated Dose 28-day Oral Toxicity Study in Rodents".

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

other: OFA

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA Credo
- Age at study initiation: 8 weeks
- Weight at study initiation: males 249-334 g; females: 177-232 g
- Fasting period before study: did not
- Housing: wire meshed bottom stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): free assess to tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+-2C
- Humidity (%):40-70
- Air changes (per hr):10-11
- Photoperiod (hrs dark / hrs light):12/24

IN-LIFE DATES: From: December 26 To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: suspension of 0.6% methylcellulose aqueous solution

Details on oral exposure:

Method of administration:
Gavage

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Test duration: 35 days.

also 2 week reversibility study conducted from day 36 to day51

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg
Basis:
other: gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
62.5 mg/kg
Basis:
other: gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg
Basis:
other: gavage

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg
Basis:
other: gavage

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day
Male: 5 animals at 62.5 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 62.5 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

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):random
-
A 2 week reversibility study was conducted from days D36 to D51 where 5 males and 5 females from the control and high dose groups were observed.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily on treatment days

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: prior to exposure and then once a week

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment and then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: D30 and day 50
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes for 16+2 hours
- How many animals:all animals
- Parameters checked in table [No.?] were examined. yes

URINALYSIS: Yes
- Time schedule for collection of urine: day 30 and day 50
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes 18 hours
- Parameters checked : voluem.color and clearness and multi-reagent strips

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: day 26 males, day 27 females, motor activity day 28
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / other: all

OTHER: blood chemistry and coagulation, mortality

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
macroscpic, organ weight and microscopic-standard organs and tissues
HISTOPATHOLOGY: Yes
--organs and tissues - microscopic, bone marrow smear

Statistics:

parametric methods:
step 1: homogeneity of intergroup vairance
step 2: equality of intergroup means
step 3: pair wise comparison of means between the treated groupd and the control groups

non-parametric methods:
1). equality of intergroup means
2). pair wise comparison of means between the treated groupd and the control groups

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS pyylism at >= 62.5 one female, solied urogenital area( only one female at 250 mg/kg

AND MORTALITY:

BODY WEIGHT AND WEIGHT GAIN : low body weight gain during first week of treatment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): not studied

FOOD EFFICIENCY: not studied

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): not studied

OPHTHALMOSCOPIC EXAMINATION:not studied
HAEMATOLOGY:at 250 mg/kg slightly increased PCV, MCHC and MCV; at 1000 slightly increased hemoglobin levels in females

CLINICAL CHEMISTRY: coagulation no treatment effects; >=62.5 slightly increased potassium levels (females); >= 250 mg/kg slight increased ALAT activity females; at 1000 mg/kg slightly increased creatinine levels(males), total cholestrol(females) & calcium levels (females )

URINALYSIS: at >-250 mg/kg slightly increased specifc gravity ( males) at 1000 mg/kg slightly decreased pH, slightly increased urinary volume and presence of leukocytes and proteins( males)

NEUROBEHAVIOUR: at 1000 mg/kg decreased spontaneous activity in females on day 12,19,26 and 40; and decreased motor activity in females

ORGAN WEIGHTS at >- 62.5 increased absolute relative liver weight(females only); >- 250 increased relative kidney weight in males

GROSS PATHOLOGY: no treatment related changes

HISTOPATHOLOGY: at >- 62.5 very slight or slight hypertrophy of centrilobular hepatocytes ( males only); >- 250 kidney: increased number of hyalin droplets in the proximal tubular cells in males

HISTOPATHOLOGY: NEOPLASTIC (if applicable): at >-250 mg/kg very slight or slight hypertrophy of centrilobular hepatocytes in malesand at 1000 mg/kg kidneys inreased number of hyalin droplets in the proximal tubular cells in males.

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS: animals found dead on D5- dehydration, rectal prolapse blood on the limbs and the muzzle

Effect levels

Dose descriptor:

NOAEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased absolute and/ot relative liver weights (females), very slight or slight hypertrophy of centrilobular hepatocytes ( males only),slightly increased potassium levels (females only), ptylism ( only one female)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

For the reversibility study:

D50: all clinical signes were reversible; no treatment related effects for hematology and for blood chemistry.

necropsy:

macrsopy: no treatement related chnges.

organ weight - increased relative liver wieghts in males and females

microsopy- kidneys hyalin droplets in the proximal tubular cells in males.

Applicant's summary and conclusion

Conclusions:

NOAEL 62.5 mg/kg bw/day(nominal).