Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 January 2006 - 28 September 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Well documented GLP study performed according to OECD Guideline 422 and EPA/OPPTS 870.3650, on the hydrochloride salt of the submission substance. Erroneously, several females of all test groups including the controls were gavaged with application volumes being 0.1 -0.3 mL above or below the target volumes on two days. These marginal and transient deviations from the study protocol are not considered to have any influence on the outcome of this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
dosing volumes (see field 'Any other information on materials and methods incl. tables)
Qualifier:
according to
Guideline:
other: EPA/OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
yes
Remarks:
dosing volumes (see field 'Any other information on materials and methods incl. tables)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hydrochloride salt of 3-Methoxypropylamine
- Molecular formula (if other than submission substance): C4 H12 Cl N O
- Molecular weight (if other than submission substance): 125.6 g/mole
- Substance type: Aqueous solution / liquid, orange, clear
- Physical state: Aqueous solution/liquid
- Lot/batch No.: BA 1464
- Storage conditions: Room temperature
- Other:
Test substance No.: 05/0675-1
Concentration of stock solution: 67.2 g 3-Methoxypropylamine hydrochloride per 100 g aqueous solution

Specific details on test material used for the study:
- Name of test material (as cited in study report): Hydrochloride salt of 3-Methoxypropylamine
- Molecular formula (if other than submission substance): C4 H12 Cl N O
- Molecular weight (if other than submission substance): 125.6 g/mole
- Substance type: Aqueous solution / liquid, orange, clear
- Physical state: Aqueous solution/liquid
- Lot/batch No.: BA 1464
- Storage conditions: Room temperature
- Other:
Test substance No.: 05/0675-1
Concentration of stock solution: 67.2 g 3-Methoxypropylamine hydrochloride per 100 g aqueous solution

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Crl:WI(Han)
- Age at study initiation: 11 - 13 weeks
- Weight at study initiation: male animals (264.0 g) and female animals (200.2 g)
- Fasting period before study: food and water were available ad libitum except during the fasting period (16 to 20 hours prior to necropsy) and measurement of motor activity)
- Housing: The rats were housed individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2), with following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until sacrifice, the pregnant animals and their litters were housed in Makrolon type M III cages (floor area about 800 cm2). The M III cages were also supplied by Becker & Co. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy. The cages with the test animals were arranged on the racks in such way that uniform experimental conditions (ventilation and light) were ensured. Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, Germany (floor area about 800 cm2) and small amounts of absorbant material.
- Diet (e.g. ad libitum): The food used was ground Kliba maintenance diet mouse/rat "GLP", supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): from water bottles available ad libitum (except during the fasting period and measurement of motor activity).
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For preparation of the administration solutions, the test substance was weighed in a graduated measuring flask (corresponding to the dose group), topped up with tap water and dissolved by shaking.

The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals that took into account the stability of the test substance preparation.

The pH-value of each test substance preparation was measured before dosing. All dosing solutions were in the pH range of 6.0 to 7.5.
Details on mating procedure:
Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner about 4.00 p.m. until 7.00-9.00 a.m. 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 "day 0" and the following day "day 1" post coitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical studies were carried out in compliance with GLP.
Method: Potentiometric titration
Discussion of results: Assuming a content of the test item of 100% at 0h, at least approximately 94% of the test item were stable in the vehicle for at least 11 days at ambient temperature.
Duration of treatment / exposure:
The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period and 4 days of lactation in females.
Frequency of treatment:
Daily at the same time in the morning (exception: no administration to animals being in labor).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The oral route was selected as it is considered to be the route whereby the majority of exposure would occur.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- At least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during "handling", fur, skin, salivation, nose discharge, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
- In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning).
The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (day 0 post coitum) and on days 7, 14 and 20 post coitum.
- Females with litter were weighed on the day of parturition (day 0 post partum) and on day 4 post partum.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
Food consumption was determined once a week (in a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period
- Food consumption of the F0 females with evidence of sperm was determined on days 0, 7, 14 and 20 post coitum
- Food consumption of F0 females, which gave birth to a litter was determined on days 0 and 4 post partum.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

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

Other: 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 (1964). Then, the respective uteri were rinsed carefully with fresh tap water. The implantation sites were recorded for the calculation of the postimplantation loss.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
Pup numbers and status at delivery:
all pups delivered from the F0 partents 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 onec 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 (day 0 post partum) and pups dying between days 1-4 of 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 lactation day 4. Furthermore the viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of liveborn pups on the day of birth) x 100
Sex ratio:
On the day of birth (day 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.
The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100
Pup 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 (day 1 post partum) and on day 4 after birth.
Pups' body weight change was calculated from these results.
Furthermore the body weights on day 1 post partum 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):
Necropsy: The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
Organ weights: the weights of the following organs were determined in all animals sacrificed at scheduled dates: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart
Organ/Tissue fixation: the following organs or tissues were fixed in 4% formaldehyde solution: all gross lesions, brain, spinal cord (cervical, thoracic and lumbar cord), sciatic nerve, pituitary gland, salivary glands, thyroid glands, parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes (axillar and mesenteric), spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), eyes with optic nerve, femur with knee joint, skin and skeletal muscle.
Histopathology: all gross lesions, trachea, lungs, liver, kidneys, spleen, adrenal glands, heart, brain, spinal cord (cervical, thoracic, lumbar), sciatic nerve, thyroid glands/parathyroid glands, testes, epididymides, ovaries, oviducts, uterus, vagina, prostate glands, seminal vesicles, coagulation glands, thymus, lymph nodes, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, bone marrow.
Postmortem examinations (offspring):
All surviving pups were sacrificed (by means of CO2) on day 4 p.p., were examined externally, eviscerated and their organs were assessed macroscopically. Stillborn pups and all pups died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups were discarded after their evaluation.
Statistics:
Food consumption, body weight and body weight change, number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss: Dunnet's test (two-sided) for the hypothesis of equal means)
Male and female mating index, male and female fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters, with affected pups at necropsy: Pairwise comparison of each dose group with the control group using Fisher's exact test for the hypothesis of equal proportions
Proportions of affected pups per litter with necropsy observations: pairwise comparison of each dose group with the control group using the Wilcoxon-test (on-sided) for the hypothesis of equal medians
Feces, rearing, grip strength of forelimbs and hindlimbs, landing footsplay test, motor activity: non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
Clinical pathology parameters, except reticulocytes and differential blood count: non-parametric one-way analysis using Kruskal-Wallis test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using wilcoxon-test (two-sided) for the equal medians.
Urinalysis, except volume, color, turbidity and specific gravity: pairwise comparison of each dose group with the control group using Fisher's exact test for the hypothesis of equal proportions
Weight of anesthesized animals and absolute and relative organ weights: Kruskal-Wallis and Wilcoxon test
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 according to the following formulas:
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
*: defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
*: defined by a female with implants in utero

Female mating index (%) = (number of females mated*/number of females placed with males) x 100
*: defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant*/number of females mated **) x 100
*: defined as the number of females with implants in utero
**: defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
*: defined as the number of females with implants in utero

Offspring viability indices:
The total number of pups delivered and the number of liveborn and stillborn pups were noted and the live birth index was calculated according to the following formulas:
Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Moreover, after sacrifice of the female animals, the implantation sites were counted and the post implantation loss was calculated for each individual pregnant animal according to the following formula:
Post implantation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical observations for signs of general toxicity revealed post-dose salivation in 5 high dose males and all high dose females. Moreover, urine discoloration was observed for all mid and high dose rats. Both findings are considered to be without toxicological relevance and are, if seen in isolation, not assessed as being adverse.
Dermal irritation (if dermal study):
not examined
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced urine volumes with subsequently increased specific gravity were excreted by female rats of the top dose group (1000 mg/kg body weight/day). It is likely, that this was caused by a decreased water intake. It is concluded that the changes in urinalysis are not caused by a direct toxic effect of the test compound on the kidney and are not considered adverse in nature. This assessment is supported by the fact, that kidney weights as well as gross and histopathological evaluations of this organ did not give any indications for substance-induced alterations in the high dose females.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Clinical pathology examinations suggested a slightly impaired liver function of the high dose females. The production of albumin and some other proteins, which are synthesized in the liver cells, was probably decreased. Additionally, the ability of the hepatocytes to eliminate bilirubin from the body by conjugation was probably diminished. Moreover, higher triglyceride levels were found in the blood of these dams. The signs indicating a disturbed liver function are considered as adverse substance-induced effects. However, this original finding is challenged. Taking into account all the other information available on this study (no histopathological correlation as liver observations are similar to the controls), these changes seem the result of an adaptive process rather than an adverse effect. This assumption is also supported by the fact that no changes have been observed in ALT, AST, ALP and GCP levels.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at 1000 mg/kg bw/d in F0 parental animals.
Key result
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
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:
no effects observed

Details on results (F1)

The number of delivered F1 pups/litter, their postnatal survival and their body weights remained unaffected by administration of the test substance. Clinical and/or gross necropsy examinations of the F1 pups revealed only findings, which were considered to be spontaneous in nature.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects at 1000 mg/kg bw/d in F1 pups.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Parental animals

Blood chemistry (mean+/-SD (number of animals)) - statistical method: Kruskal-Wallis + Wilcoxon-test, Two sides, *p<0.05, ** p<0.010

 

 Male         

Female          

   0 mg/kg bw  100 mg/kg bw  300 mg/kg bw  1000 mg/kg bw  0 mg/kg bw  100 mg/kg bw  300 mg/kg bw  1000 mg/kg bw
 ALT µkat/l  0.49 +/-0.10 (5)  0.52 +/-0.09 (5)  0.49 +/-0.06 (5) 0.51 +/-0.11 (5)   0.46 +/-0.24 (5)  0.31 +/-0.07 (5)  0.30 +/-0.05 (5)  0.38 +/-0.09 (5)
 AST µkat/l  1.31 +/-0.30 (5)  1.46 +/-0.22 (5)  1.42 +/-0.23 (5)  1.32 +/-0.11 (5)  1.52 +/-0.18 (5)  1.38 +/-0.31 (5)  1.19 +/-0.20 (5)  1.20 +/-0.19 (5)
 ALP µkat/l  1.52 +/-0.27 (5)  1.46 +/-0.17 (5)  1.32 +/-0.17 (5)  1.53 +/-0.10 (5)  0.57 +/-0.16 (5)  1.66 +/-2.14 (5)  0.58 +/-0.07 (5)  0.71 +/-0.20 (5)
 SGGT nkat/l  0 +/-0 (5)

 0 +/-0 (5)

 0 +/-0 (5)  0 +/-0 (5)  0 +/-0 (5)  19 +/-42 (5)  0 +/-0 (5)  0 +/-0 (5)
 INP mmol/l  1.83 +/-0.06 (5)  1.91 +/-0.18 (5)  1.88 +/-0.06 (5)  2.06 +/-0.08** (5)  1.55 +/-0.15 (5)  1.64 +/-0.12 (5)  1.48 +/-0.17 (5)  1.74 +/-0.15 (5)
 Bilirubine µmol/l  1.96 +/-0.45 (5)  2.04 +/-0.23 (5)  2.13 +/-0.47 (5)  2.70 +/-0.62 (5)  2.51 +/-0.29 (5)  2.88 +/-0.28 (5)  2.76 +/-0.39 (5)  3.18 +/-0.43* (5)
 Protein 64.80 +/-1.86 (5)   63.86 +/-1.61 (5)  62.99 +/-2.20 (5)  62.54 +/-0.78 (5)  69.39 +/-1.43 (5)  67.35 +/-4.27 (5)  68.07 +/-1.71 (5)  64.12 +/-1.53** (5)
 Albumin  37.65 +/-0.98 (5)  36.59 +/-0.58 (5)  36.82 +/-1.41 (5)  36.57 +/-0.61 (5)  40.96 +/-0.87 (5)  38.99 +/-3.73 (5)  40.10 +/-0.77 (5)  37.50 +/-0.85** (5)
 Globulin  27.15 +/-1.04 (5)  27.27 +/-1.07 (5)  26.17 +/-1.48 (5)  25.97 +/-0.76 (5) 28.43 +/-1.10 (5)   28.53 +/-1.06 (5)  27.97 +/-1.07 (5)  26.62 +/-1.30 (5)
 Triglycerides  0.66 +/-0.45 (5)  0.52 +/-0.16 (5)  0.58 +/-0.34 (5)  0.55 +/-0.18 (5)  0.32 +/-0.05 (5)  0.32 +/-0.04 (5)  0.43 +/-0.10 (5)  0.42 +/-0.06* (5)
 Cholesterol  1.40 +/-0.19 (5)  1.74 +/-0.36* (5)  1.72 +/-0.14* (5)  1.47 +/-0.18 (5)  1.49 +/-0.22 (5)  1.55 +/-0.18 (5)  1.61 +/-0.32 (5)  1.57 +/-0.33 (5)

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive performance and fertility is 1000 mg/kg body weight/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance is 1000 mg/kg body weight/day for the F0 generation. The NOAEL for developmental toxicity is 1000 mg/kg bw/d. The NOAEL for developmental toxicity in the F1 progeny of the test substance treated groups was found to be 1000 mg/kg body weight/day.