N-[3-(dimethylamino)propyl]oleamide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to OECD guideline 421 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]oleamide

EC Number:

203-661-5

EC Name:

N-[3-(dimethylamino)propyl]oleamide

Cas Number:

109-28-4

Molecular formula:

C23H46N2O

IUPAC Name:

(9Z)-N-[3-(dimethylamino)propyl]octadec-9-enamide

Test material form:

other: liquid

Details on test material:

Test substance no.: 07/0589-1
Batch: R401/57
Purity: 78.2 area-%
Color: reddish
Homogeneity: given (visible)
Storage stability: The storage stability under storage conditions was confirmed by reanalysis.
Storage conditions: ambient (room temperature); under light exclusion and covered with N2.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Strain: Crl:WI(Han)
- Sex: males and females
- Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at start of administration period: 11-12 weeks
- Housing: individually in Makrolon cages, type M III, supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2), in fully air-conditioned rooms (20-24 °C) with 30-70% relative humidity and 10 air changes per hour.
- Light/Dark cycle: 12h/12h
- Diet: 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).
- Acclimatization: 6 days after arrival in the facility

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

drinking water

Details on exposure:

- Test substance preparation: appropriate amount of test substance was weighed out depending on desired concentration and filled up with vehicle to the desired volume. The test-substance preparations were prepared in such intervals that the stability was guaranteed.
- Daily administered volume: 10 ml/kg bw

Details on mating procedure:

- Male/female ratio per cage: 1:1
- Mating period: overnight for a maximum of 2 weeks
- Proof of pregnance: sperm in vaginal smear

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water for a period of 7 days was proven before the start of the administration period.

Duration of treatment / exposure:

The duration of treatment covered premating period of 2 weeks and a mating period (max. of 2 weeks) in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Frequency of treatment:

Once daily.

Details on study schedule:

- Beginning of mating of F0 animals: at least 13 days after beginning of treatment
- Duration of mating period: max. 2 weeks
- Timepoints of sacrifice (F0 animals): study day 29 for males, study day 53 for females, after a fasting period for at least 16-20 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 75 and 200 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection: Dose levels were selected based on the results of the OECD 407 study (see section 7.5.1 of the IUCLID).

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Conducted daily before and about 1 hour after applications for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Littering and lactation behvior of the dams was generally evaluated in the mornings in combination with the daily inspection of the dams.

BODY WEIGHT:
Determined once a week with the following exceptions:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
- Females without a litter were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Determined once a week, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and 4.

Oestrous cyclicity (parental animals):

not examined

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).

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.

PUP VIABILITY/MORTALIY
Checked twice daily on workdays and once daily on weekends or holidays. Pup viability was determined via the viability index (see section "offspring viability indices)

SEX RATIO
Determined on the day of birth by observing the anogenital distance. The sex ratio was calculated at PND 0 and 4 according to the following formulat: Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) * 100

CLINICAL OBSERVATION
Performed daily

BODY WEIGHT
Pups were weighed one day and four days after birth. Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which
weighed less than 25% of 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).

PUP NECROPSY OBSERVATIONS
All surviving pups (after sacrifice on PND 4), all stillborn pups and those pups, which died ahead of 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.

Postmortem examinations (parental animals):

BODY/ORGAN WEIGHTS:
Anesthetized animals, testes, epididymides, ovaries

ORGAN/TISSUE FIXATION:
all gross lesions, pituitary gland, prostate gland, seminal vesicles with coagulation glands, uterus, oviducts, cervix uteri, vagina, left testis, left epididymis, ovaries.

HISTOPATHOLOGY:
All gross lesions, left testis, left epididymis, ovaries

Postmortem examinations (offspring):

After necropsy, pups were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

CLINICAL EXAMINATIONS:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter, implantation sites, post implantation loss: Simultaneous comparison of all dose groups with the control group using the DUNNETT-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
- Males with > 4% abnormal sperm (90%-Quantile of the control group is equal to 4%): FISHER'S EXACT test
- Total spermatids/g testis, total sperm/g cauda epididymides: Pairwise comparison of the dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
- Sperm motility (%): Pairwise comparison of the dose group with the control group using the WILCOXON-test (one-sided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians
- Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

PATHOLOGY:
- Weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS 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 the WILCOXON test for the hypothesis of equal medians

Reproductive indices:

MALES:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) * 100 (confirmed mating was 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) * 100 (proven fertility was defined by a female with implants in utero)

FEMALES:
- Female mating index (%) = (number of females mated / number of females placed with males) * 100 (Mated females were 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) * 100 (prenant females were defined as the number of females with implants in utero; mated females were 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 prengant) * 100 ((prenant females were defined as the number of females with implants in utero)
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) * 100
- Post implantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) * 100

Offspring viability indices:

Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on the day of birth) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see "details on results (parental animals)"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see "details on results (parental animals)"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see "details on results (parental animals)"

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see "details on results (parental animals)"

Details on results (P0)

MORTALITY:
No test substance related mortalities occurred in any test group.

CLINICAL SIGNS (except gestation and lactation periods)(see also tables IA-001/002/003/004 in section "attached background material"):
200 mg/kg dose group:
In 6 male animals, salivation after treatment was observed starting after 1 week of treatment. After 2 weeks of treatment salivation before treatment was seen in 5 male animals.
In addition, respiration sounds were observed predominantly after treatment in 3 male animals temporarilty during the administration period.
Eyelid halfclosure was also observed in 1 male, although only in week 0.
In all females of the 200 mg/kg bw/d group, salivation was observed most of the time after treatment during premating. Respiration sounds (predominantly after treatment) were observed in 5 animals, occasionally during premating. These findings were assessed as being related to treatment.

75 mg/kg dose group:
1 male showed only salivation before treatment in the third week of treatment. This finding was rather caused by conditioning due to treatment by gavage than reflecting toxicity or an adverse finding.

25 mg/kg dose group:
Alopecia was observed in one female in the seventh week of treatment. This single finding was clearly incidental and not related to the test substance.

CINICAL OBSERVATIONS (females during gestation) (see also tables IA-005/006 in section "attached background material").
200 mg/kg dose group:
Salivation predominantly after treatment was observed in all females most of the time during the gestation period. Respiration sounds predominantly after treatment were observed in 3 females during gestation. Temporarily during gestation eyelid half closure (2 animals) and piloerection (1 animal) were observed. These findings were assessed as being related to treatment.

75 mg/kg dose group:
Respiration sounds after treatment were observed in 1 animal and only on gestation days 3 and 4. These two single occurrences were clearly without any toxicologically relevance and, therefore, assessed as being not related to treatment.

CLINICAL OBSERVATIONS (females during lactation) (see also tables IA-007/008 in section "attached background material"):
In test group 3 (200 mg/kg bw/d) predominantly salivation after treatment was observed in all females at the most time during the lactation period. Respiration sounds predominantly after treatment were observed in 2 females during lactation. Eyelid half closure and piloerection was observed in each one female animal temporarily during lactation. These findings were assessed as being related to treatment.

FOOD CONSUMPTION:
Food consumption of male and female animals of all treatment groups was comparable to that of the controls during the entire study period.

BODY WEIGHTS (see also tables IA-013/014/015/016/017/018/019/020 in section "attached background material"):
Body weight change of male animals of the 200 mg/kg bw/d group was significantly decreased in the first week of treatment (47% less) as, in addition, the overall body weight gain from week 0 to week 4 was significantly decreased (20% less). This finding was assessed as related to treatment and caused by an incipient general systemic toxicity. Body weight change values of male animals of test substance-treated groups 1 and 2 (25 and 75 mg/kg bw/d) and of female animals of test groups 1, 2 and 3 (25, 75 and 200 mg/kg bw/d) were generally comparable to that of the concurrent control group during premating, gestation and lactation periods.


REPRODUCTIVE PERFORMANCE (see also tables IA-021/023/024 in section "attached background material"):
- male mating index: 100% in all test groups incl. controls
- male fertility indices: decreased to 80% in the 200 mg/kg bw/d group as compared to 100% in the lower dose groups. One sperm positive female of test group 1 (25 mg/kg bw/d), two sperm positive females of test group 2 (75 mg/kg bw/d) and two sperm positive females of test group 3 (200 mg/kg bw/d) did not deliver any F1 pups. The female rat from the 25 mg/kg group and one of the females in the 75 mg/kg groups, however, had implantation sites in utero indicating their fertility. Gross and histopathological examinations of male animals and their female mating partners failed to show a relevant morphological correlate for the apparently impaired fertility.
- female mating index: 100% in all test groups incl. controls
- female fertility index: varied between 80% in test group 3 (200 mg/kg bw/d) and 100% in test groups 0 and 1 (0 and 25 mg/kg bw/d). Gross and
histopathological examinations failed to show a relevant morphological correlate for the apparently impaired fertility
- duration of gestation: significantly extended in test group 2 (22.4 days; 75 mg/kg bw/d) when compared to the control (21.9 days) whereas test groups 1 (25 mg/kg bw/d) and 3 (200 mg/kg bw/d) showed no significant differences. With regard to the fact that the mean duration of gestation in test group 2 (75 mg/kg bw/d) was only slightly outside the range of the historical control data (21.5-22.3 days) and no significant changes were noted in test group 3 (200 mg/kg bw/d) the significant difference in test group 2 (75 mg/kg bw/d) was assessed as being spontaneous in nature and not test substance-related.
- gestation index: varied between 89% in test group 2 (75 mg/kg bw/d) and 100% in test groups 0 and 3 (0 and 200 mg/kg bw/d). One low-dose F0 parental female and one mid-dose F0 parental female which were sperm positive, showed implantation sites at necropsy but delivered no pups. The mean number of implantation sites including the controls was 13.6, 12.4, 11.3 and 12.0 implants/dam in test groups 0-3 (0, 25, 75 and 200 mg/kg bw/d).
- Postimplantation loss: significantly increased in test group 2 (75 mg/kg bw/d). Due to the fact that no increase was noted in test group 3 (200 mg/kg bw/d) the finding was assessed as being spontaneous in nature and not test substance-related. The apparently high average postimplantation loss (expressed in %) in test groups 1 and 2 (25 and 75 mg/kg bw/d) was a consequence of the calculation method rather than a real effect and related to the total loss (postimplantation loss set to 100%) of both implants in female 119 (test group 1, 25 mg/kg bw/d) and of the single implant in female No. 125 (test group 2, 75 mg/kg bw/d). Excluding these animals from the mean would result in values of 6.3% (test group 1, 25 mg/kg bw/d) and 12.5% (test group 2, 75 mg/kg bw/d). In this case, only the latter value would exceed the range of the historical control data. However, a dose-response relationship was not observed with regard to test group 3 (200 mg/kg bw/d).
- live birth indices: ranging between 99% and 100%. Only one stillborn pup was seen in test group 3 (200 mg/kg bw/d). This incidental finding was clearly within the range of the historical control data and not related to treatment.

SPERM PARAMETERS (see also table IA-022 in section "attached background material"):
No treatment-related effects were noted for the sperm parameters, examined at or after the sacrifice of the F0 parental males in all test groups.

PATHOLOGY (see also tables IB-1-6 in section "attached background material"):
Compared to the control (set to 100%) the terminal body weights of male F0 animals were significantly decreased to 96% in test group 2 (75 mg/kg bw/d) and to 95% in test group 3 (200 mg/kg bw/d). A treatment-related weight decrease in males could not be excluded. All other mean absolute weight parameters did not show relevant differences compared to the control group and were regarded to be within the normal biological range of test animals of that age.
Compared to controls (set to 100%) no deviations in relative sex organ weights were recorded for test groups 1-3 (25, 75 and 200 mg/kg bw/d).
All gross lesions observed in test animals occurred singularly. They were considered to be spontaneous lesions in origin and not related to treatment.
All noted findings were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them were considered to be incidental and/or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Reproductive performance and fertility

2

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Sex:

male

Basis for effect level:

other: General systemic toxicity, based on impaired body weight

3

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Sex:

female

Basis for effect level:

other: General systemic toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

see "details on results (offspring)"

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)

For litter data see also tables IA-025-027 in section "attached background material".

VIABILITY:
A significant increase of pups that died during lactation was observed for test group 3 (200 mg/kg bw/d) and a relation to treatment was assumed. The viability index as indicator for pup mortality between PND 0-4 varied between 93% (test group 3, 200 mg/kg bw/d) and 99% (test group 0, control). In test group 1 and 2 (25 and 75 mg/kg bw/d) no test substancerelated changes were obtained.

SEX RATION:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show biologically relevant differences between controls and test groups.

CLINICAL OBSERVATIONS:
The F1 pups did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (see also tables IA-028/029 in section "attached background material"):
Mean pup body weights and pup body weight changes in the test substance-treated groups 1-3 (25, 75 and 200 mg/kg bw/d) were comparable to the concurrent control values. The observable differences between the test groups were assessed as being spontaneous in nature and without any biological relevance. The numbers of runts were 1, 0, 0 and 7 in test groups 0-3 (0, 25, 75 and 200 mg/kg bw/d). All 7 runts of test group 3 (200 mg/kg bw/d) belonged to one litter. Since runts appeared only this individual litter, a relation to dosing was not assumed.

NECROPSY (see also table IA-030 in section "attached background material"):
No gross findings were observed during pup necropsy in any test group.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

75 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Developmental toxicity, based on decreased viability index at 200 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

It can be concluded that under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility in male and female Wistar rats was 75 mg/kg bw/d. The NOAEL for general, systemic toxicity of the test substance was 25 mg/kg bw/d for the F0 parental males based on impaired body weight data and 200 mg/kg bw/d for females. The NOAEL for developmental toxicity was 75 mg/kg bw/d for both sexes as the viability index was decreased at a dose level of 200 mg/kg bw/d.

Executive summary:

3-Dimethyl-aminopropyl-ölsäureamide was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at doses of 0, 25, 75 and 200 mg/kg bw/d in order to observe the possible effects of the test substance on the integrity and performance of the reproductive system in both sexes.

Regarding clinical examinations, salivation was observed predominantly after treatment in both sexes of test groups 2 (75 mg/kg bw/d) and 3 (200 mg/kg bw/d). From the temporary, short appearance immediately after dosing salivation was most likely induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. In addition, respiration sounds occurred in several animals during different phases of the study and not throughout the entire study period. This finding was also assessed as being related to treatment and possibly associated to the administration procedure. However, gross lesions were not observed during gross pathological examination. The viability index as indicator for pup mortality was significantly decreased in test group 3 (200 mg/kg bw/d). A relation to treatment was assumed although gross necropsy revealed no relevant findings. Fertility indices for male and female animals were most likely impaired by test-substance administration dose levels of 200 mg/kg bw/d and not within the historical control data. However, histopathological correlates, which could explain infertility, were not found in the sex organs of the infertile mating pairs. Regarding pathology the test substance led to no pathological findings in the parental Wistar rats, but a treatment-related decrease of absolute terminal body weights in male animals of test groups 2 (75 mg/kg bw/d) and 3 (200 mg/kg bw/d) could not be excluded definitely.

Based on the results of this study, the following dose descriptors were derived:

- NOAEL for reproductive performance and fertility in male and female Wistar rats: 75 mg/kg bw/d

- NOAEL for general, systemic toxicity of the test substance: 25 mg/kg bw/d for the F0 parental males based on impaired body weight data and 200 mg/kg bw/d for females

- NOAEL for developmental toxicity: 75 mg/kg bw/d for both sexes as the viability index was decreased at a dose level of 200 mg/kg bw/d.