1-[(2-butyloctyl)oxy]-3-(3,4-dihydroisoquinolinium-2-yl)propan-2-yl sulfate

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 2006-11-15 to 2006-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

July 27, 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

96/54/EEC

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japan/MHW: Guidelines for Toxicity Testing of Chemicals, twenty-eight-day repeated dose toxicity in mammalian species ; MITI/MHW, 1987 (Translation), pp . 183-188

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sokalan PG B62;

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar rats CrI :WI(Han) were supplied by Charles River, Sulzfeld, Germany, at an age of 36 +/- 1 day. Only animals were applied free from clinical signs of disease at the start of the study .
The rats were housed singly in type DK III stainless steel wire mesh cages (floor area about 800 cm2) . Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dust free embedding). Motor activity measurements were conducted in Polycarbonate cages with wire covers (floor area about 800 cm2) and small amounts of absorbent material. The animals were housed in a fully air-conditioned room. Central airconditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity. The day/night cycle was 12 hours (12 hours light from 06 .00 a .m. - 06 .00 p.m. , 12 hours dark from 06 .00 p.m . - 06 .00 a .m.). Deviations from these ranges did not occur. The animal room was completely disinfected prior to the study using a disinfector ("AUTEX", fully automatic, formalin-ammonia-based terminal disinfector). The floor and the walls were cleaned once a week. The cleansing liquid used was water containing about 0 .1 % Incidin.
The food used was ground Kliba maintenance diet mouse/rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

The test substance was administered daily by gavage over 4 weeks. Control animals received drinking water with 0.5% carboxymethylcellulose, only .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF Aktiengesellschaft, Ludwigshafen, Germany .
The stability of the test substance in 0 .5% Carboxymethylcellulose in doubly distilleed water over a period of 4 days at room temperature was proven before the start of the study (Project No. 01 Y0655/058015).
Before the beginning of the administration period, 3 samples were taken from the lowest and highest concentration for homogeneity analysis; these samples were used as a concentration control at the same time. Only one sample was taken from the intermediate dose for concentration control. A reserve sample of each sample was removed .

Duration of treatment / exposure:

28 days (treatment)

Frequency of treatment:

daily, 7 days a week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw in 0.5% CMC-solution
Basis:

No. of animals per sex per dose:

Control and high dose groups consisted of each 10 animals per sex (5 in the main groups, 5 in the recovery groups), whereas low and mid dose groups consisted of each 5 animals per sex (main groups).

Control animals:

yes, concurrent vehicle

Details on study design:

At the end of the administration period the animals of the main groups were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours . The animals of the recovery groups were maintained for another two weeks with ground diet. All surviving animals were sacrificed after a fasting period (withdrawal of food) for at least 16- 20 hours.

Positive control:

No positive control.

Examinations

Observations and examinations performed and frequency:

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.
Functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the treatment period. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. At the end of the study all animals were assessed by gross pathology, followed by histopathological examinations.

Sacrifice and pathology:

After 4 weeks of treatment 5 animals per sex of all dose groups were sacrificed. The remaining 5 animals per sex
of control and high dose groups were maintained for another 14 days without administration of the test substance.

Other examinations:

No other examinations.

Statistics:

Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed according to the following tests:
- DUNNETT's test
- KRUSKAL-WALLIS test
- Welch t-test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

No substance-related adverse findings obtained during administration period and during recovery period in any of the treatment groups.

Mortality
No animal died prematurely during the present study.

Clinical examinations
Slight salivation after treatment was observed in 3 high dosed animals of either sex on several days of the study. This isolated clinical finding without any correlative to other examinations within the entire study was most likely caused by a bad taste of the test article than reflecting an adverse effect.

Food consumption
A statistically significant increase of food consumption was measured in females of group 2 on days 7, 14 and 28 . These findings only observed in mid dosed females without any doseresponse relationship were clearly incidental and not related to treatment with the test article.

Water consumption
No substance-related overt changes in water consumption were observed.

Body weight data
No substance-related findings were observed.

Food efficiency
No substance-related findings were calculated.

Functional observational battery
Deviations from "zero values" were obtained in several animals . However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Motor activity measurement (MA)
Regarding the overall motor activity (summation of all intervals) no substance-related findings were observed in both sexes. Comparing the single intervals with the control group, the motor activity was statistically significantly decreased in females of group 3 at interval 1, only. This single finding without any influence on the overall motor activity was clearly not related to the administration of the test article.

Hematology
There are no treatment-related changes in the hematological parameters measured.

Clinical chemistry
Compound-related differences in clinical chemistry parameters were not evident at any dose
level in either males or females.

Urinalysis
No test substance-related effects were observed in urinalyses of either sex.

Absolute weights
In males, the thymus weight was slightly but significantly decreased in the mid dose group. This decrease is considered to be incidental and of no biological significance (no doseresponse relationship, no histomorphological correlate). In females, the spleen weight of the high dose group was significantly decreased. As there is no histomorphological correlate that would explain the weight decrease, this decrease is as well considered to be incidental and of no biological significance . In the recovery groups, no weight changes were noted.

Relative Organ Weights
In males, the thymus weight was slightly but significantly decreased in the mid dose group. This decrease is considered to be incidental and of no biological significance (no doseresponse relationship, no histomorphological correlate). In females, the spleen weight of the high dose group was significantly decreased . As there is no histomorphological correlate that would explain the weight decrease, this decrease is as well considered to be incidental and of no biological significance. In the recovery groups, no weight changes were noted.

Gross lesions
All gross lesions noted are of spontaneous origin and not related to treatment.

Histopathology
All findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment. One moderate and one severe case of a chronic nephropathy (CPN) were observed in one low and one high dose male . Those lesions as well as the single occurrence of basophilc tubules, protein casts, or focal nephritis in single control and high dose animals are regarded to be spontaneous lesions and are not related to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a 28 -day repeated dose toxicity study with 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate no substance-related adverse findings were obtained. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg body weight per day in both sexes.

Executive summary:

In a 28 -day repeated dose toxicity study, 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate was administered to male and female Wistar rats by gavage at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg body weight per day over a period of 4 weeks. Control and high dose groups consisted of each 10 animals per sex, whereas low and mid dose groups consisted of each 5 animals per sex. After 4 weeks of treatment 5 animals per sex of alt dose groups were sacrificed (main groups). The remaining 5 animals per sex of control and high dose groups were maintained for another 14 days without administration of the test substance (recovery groups). The dose volume was 5 ml/kg bw.

Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Functional observational battery (FOB) as well as measurement of motor activity (MA) was carried out at the end of the treatment period. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. At the end of the study all animals were assessed by gross pathology, followed by histopathological examinations.

No substance related effects were observed in any treatment group. Concerning Clinical Pathology no treatment-related adverse findings were measured. Moreover, regarding Pathology, there were no substance-related weight changes, gross lesions or microscopic findings in male and female Wistar rats observed after 4 weeks of treatment. In addition, during the recovery period also no effects were noted.

In conclusion, under the conditions of the present study no substance-related adverse findings were obtained. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg body weight per day in both sexes.