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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2009-09-15 to 2010-03-16

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:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

Juy 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Name of test substance: Sokalan PG B62

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test species and strain:
Wistar Rat; Crl:WI(Han)
All animals were free from clinical signs of disease at the start of the study. The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating

Reason for species selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected

Age (at supplied); sex:
10-11 weeks (males/females)

Supplier:
Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld

Identification:
The rats of the parental generation (F0 animals) were identified clearly by ear tattoo. All live pups were identified by skin tattoo on PND 1.

HOUSING AND DIET
The rats were housed individually in Makrolon cages, type M III, supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2). Bedding in the Makrolon cages was Lignocel dust free bedding supplied by SSNIFF, Soest, Germany. For enrichment, wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added. Nesting material (cellulose wadding) was provided for pregnant females toward the end of gestation. The cages with the test animals were arranged on the racks in such way that uniform experimental conditions (ventilation and light) were ensured.
The animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20 - 24°C, a range of relative humidity of 30-70% and 10 air changes per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours (12 hours light from 06:00 - 18:00 h, 12 hours dark from 18:00 - 06:00 h). 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 with water containing an appropriate disinfectant.
The food used was ground Kliba maintenance diet mouse/rat “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum (except during the fasting period).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 %

Details on exposure:

Test-substance preparations and preparation frequency
The test substance was applied as a suspension. To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (drinking water with 0.5% carboxymethylcellulose) was filled up to the desired volume, subsequently mixed using a magnetic stirrer and a high speed homogenizer and kept homogeneous during administration using a magnetic stirrer. The test-substance preparations were prepared in such intervals that the stability was guaranteed.

Details on mating procedure:

Each of the male and female animals was mated overnight in a 1:1 ratio, generally, for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16:00 h until 07:00 - 09:00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted "gestation day (GD) 0" and the following day "GD 1".

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 SE. The stability of the test substance in doubly distilled water with 0.5% carboxymethylcellulose at room temperature for a period of 4 days was verified before the start of the study in a comparable batch of the test substance (Project-No. 01Y0655/058015). Homogeneity and concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start and at the end of the administration period. Of each sample, one additional reserve sample was retained.

Duration of treatment / exposure:

Males: 31 days
Females: 49 or 56 days

Frequency of treatment:

daily

Details on study schedule:

At least 13 days after the beginning of treatment, males and females from the same test group were mated overnight in a ratio of 1:1, generally. All F0 males were sacrificed under isoflurane anesthesia on study day 31 followed by necropsy. The females were allowed to litter and rear their pups until day 4 after parturition. F0 females were sacrificed under isoflurane anesthesia on study days 49 and 56 followed by necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 100; 300 and 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. The animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer. At the start of the administration period (day 0) the rats were 11 - 12 weeks old. After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals daily, at approximately at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only (drinking water with 0.5% carboxymethylcellulose). The daily volume administered was 10 ml/kg of body weight. The calculation of the volume administered was generally based on the most recent individual body weights.

Determination of implantation sites
After sacrifice of the female animals the uterus and ovaries were removed and the implantation sites were counted. To determine the number of implantation sites in apparently non-pregnant animals, the uteri from those females were stained in 10% ammonium sulfide solution for about 5 minutes according to the method of SALEWSKI (Salewski, E.; 1964). Then, the respective uteri were rinsed carefully with fresh tap water. The implantation sites were recorded for the calculation of the postimplantation loss.

Post mortem examinations of pups
All surviving pups were sacrificed (by means of CO2) on PND 4. They were examined externally, eviscerated and their organs were assessed macroscopically. Stillborn pups and all pups died before scheduled were examined externally, eviscerated and their organs were assessed macroscopically. All pups were discarded after their evaluation.

Positive control:

no positive control

Examinations

Parental animals: Observations and examinations:

- Mortality
- Clinical observations
- Food consumption
- Body weight

Oestrous cyclicity (parental animals):

Female reproductive data were investigated.

Sperm parameters (parental animals):

Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from the F0 males of all test groups.
The following parameters were determined:
- sperm motility
- sperm morphology
- sperm head count (cauda epididymis)
- sperm head count (testis)
Sperm motility examinations and the preparation of the specimens for sperm morphology were carried out in a randomized sequence. Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and highest test group, only.

Litter observations:

Pup number and status at delivery
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and pups dying during the lactation period were determined. However, pups which died accidentally and pups which were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day of birth, and on PND 4.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. Normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups finally confirmed at necropsy.

Clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Pup body weight data
The pups were weighed one day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed more than 25% less than the mean weight of the respective control pups). The individual weights were always determined at about the same time of the day (in the morning).

Postmortem examinations (parental animals):

- Sperm parameters (see above)
- Implantation sites (see above)
- Organ weights
- Histopathology

Postmortem examinations (offspring):

All surviving pups (after sacrifice on PND 4 by means of CO2), all stillborn pups and those pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Statistical tests used:
- DUNNETT-test (two sided)
- FISHER'S EXACT test
- WILCOXON-test (one-sided)
- KRUSKALWALLIS test (two-sided)

Reproductive indices:

The pairing partners, the number of mating days until vaginal sperm was detected in female animals, and the gestational status of the females were recorded for the F0 breeding pairs. For the males, mating and fertility indices were calculated. For the females, mating, fertility and gestation indices were calculated. The total number of pups delivered and the number of liveborn and stillborn pups were noted and the live birth index was calculated. Moreover, after sacrifice of the female animals, the implantation sites were counted and the post implantation loss was calculated for each individual pregnant animal.

Offspring viability indices:

The viability index was calculated. The sex ratio was calculated at PND 0 and 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

No test substance-related, relevant findings were observed in F0 parental animals.
Regarding clinical examinations the test substance did not reveal any signs of reproductive or systemic toxicity at any dose level tested.
Regarding pathology, there were no treatment-related findings noted. All findings recorded were considered to be incidental in nature and not related to treatment.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

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

2

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

No test substance-related, relevant findings were observed in F1 pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of a reproduction/developmental toxicity screening test with 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulfate, the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1000 mg/kg bw/d for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance was also 1000 mg/kg bw/d for the F0 parental male and female animals. Treatment with the test article did not lead to any pathomorphological changes with respect to the tissue and organs examined during necropsy, organ weight determination and light microscopy. The NOAEL for developmental toxicity in the F1 progeny of the test substance-treated groups was found to be 1000 mg/kg bw/d.

Executive summary:

In a reproduction/toxicity screening test, 1-((2-Butyloctyloxymethyl)-2-(3,4-dihydro-isoquinolinium-2-yl)ethyl)sulphate was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) in order to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes. Control animals were dosed daily with the vehicle. The duration of treatment covered a two-weeks premating period and mating period in both sexes, approximately one week post-mating in males and the entire gestation period and 4 days of lactation in females.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. Food consumption of the F0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of F0 animals were determined once a week; however, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the parturition day and postnatal day (PND) 4. Pups were sexed on PND 0 and weighed one day after birth and on PND 4. Their viability was recorded until PND 4 when all pups were sacrificed with CO2 and examined macroscopically for external and visceral findings. All surviving F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Sex organ weights were recorded and a histopathological examination was performed. A detailed examination of sperm was performed.

No test substance-related, relevant findings were observed in F0 parental animals and F1 pups at all doses applied. Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was equal to the limit dose of 1000 mg/kg bw/d recommended in the respective guideline for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance was also 1000 mg/kg bw/d for the F0 parental male and female animals. Treatment with the test article did not lead to any pathomorphological changes with respect to the tissue and organs examined during necropsy, organ weight determination and light microscopy. The NOAEL for developmental toxicity in the F1 progeny of the test substance-treated groups was found to be 1000 mg/kg bw/d.