Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
07 October 2008 - 02 July 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD guideline 421 without any deviation
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 3-Dimethyl-aminopropyl-ölsäureamide
- CAS number: 109-28-4
- Physical state: Reddish liquid
- Analytical purity: 78.2 area%
Composition by GC-MS :
• cis-Octadecenoic acid, 3-dimethylaminopropyl amide 78-80 %
• trans-Octadecenoic acid, 3-dimethylaminopropyl amide 0.5 - 2 %
• C16-acid, 3-dimethylaminoproyl amide, 4 isomers 8 - 12 %
• Other carbonic acids, 3-dimethylaminopropyl amide 6-8 %
• Other carbonic acids, 3-dimethylaminopropyl amide 1-2%
- Lot/batch No.: R 401/57
- Storage condition of test material: Ambient (room temperature); under light exclusion; under Nitrogen

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, Germany
- Age at study initiation (P): 11-12 weeks
- Weight at study initiation (mean): Males (P): 315.2-319.4 g; Females (P): 206.2-209.5 g
- Housing: Housed individually in Makrolon cages, type M III, supplied by Becker & Co., Germany (floor area of about 800 cm2)
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat “GLP”, meal (Source: Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum (except during the fasting period)
- Water (e.g. ad libitum): Drinking water (from water bottles), ad libitum (except during the fasting period)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: 10 air changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 07 October 2008 To: 11 November 2008 (males) or 05 December 2008 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: PREPARATION OF DOSING SOLUTIONS: A specified amount of the test substance was weighed and mixed with water using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.25, 0.75 and 2.00 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Mated overnight for a maximum of 2 weeks
- Proof of pregnancy: Sperm in vaginal smear referred to as gestation day (GD) 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical method: Stability, homogeneity and verification of the concentrations of the test substance were analysed in separate studies (Study no.: 08L00155, 08L00306 and 08L00391) using Gas Chromatograph equipped with flame ionisation detector (FID).
- Results: Analysis results demonstrated the stability of the test substance preparations for at least 7 days at room temperature. It showed the homogeneous distribution of the test article in vehicle and the correctness of the prepared concentrations (90-110 % of the nominal concentrations).
Duration of treatment / exposure:
- Males: 2 weeks before mating, during the mating (maximum of 2 weeks) and post-mating periods (approximately 1 week)
- Females: 2 weeks before mating, during the mating period (maximum of 2 weeks), during pregnancy and 4 days of lactation
Frequency of treatment:
Once daily, 7 days/week
Details on study schedule:
Not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 75 and 200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose-levels were selected based on the results of the 28-days repeated dose toxicity study (OECD 407) (Study No. 30S0589/07105) in which the same test item was administered to Wistar rats by gavage at the dose-levels of 0, 10, 50 and 150 mg/kg bw/day. No treatment-related effects were observed at any dose level except slight signs of toxicity at 150 mg/kg bw/day as sporadic and transient salivation and/or lud breathing.
- Rationale for animal assignment: According to weight, animals were grouped randomly using a computer.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: Twice daily on working days and once daily on Saturdays, Sundays and public holidays
- Clinical observations: A cageside examination was conducted daily before and about 1 h after application for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Littering and lactation behavior of the dams was generally evaluated twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Generally, the body weight of the male and female parental (F0) animals was determined once a week. 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.

FOOD CONSUMPTION:
- Time schedule: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: food consumption was not determined in male and female F0 animals during the mating period; in females without positive evidence of sperm (during the mating period of dams used in parallel) and in females without litter (during the lactation period of dams used in parallel). Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20 and food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and 4.
Oestrous cyclicity (parental animals):
No data
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.
- 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 high-dose 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.

PUP VIABILITY/MORTALITY
- In general, a check was made for any dead or moribund pups twice daily on workdays and 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, and the viability index was calculated.

SEX RATIO
- On the day of birth (PND 0) the sex of the pups and sex ratio were determined by observing the distance between the anus and the base of the genital tubercle. The sex of the pups finally confirmed at necropsy (PND 4).

PUP CLINICAL OBSERVATIONS
- The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

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 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).
Postmortem examinations (parental animals):
SACRIFICE AND NECROPSY
- All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.
- 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.

ORGAN WEIGHTS
- Anesthetized animals were weighed and after sacrifice testes, epididymides and ovaries were weighed.

HISTOPATHOLOGY
- The following organs or tissues of the F0 generation parental animals were fixed in 4% neutral buffered formaldehyde solution: all gross lesions, pituitary gland, prostate gland, seminal vesicles with coagulation glands, uterus, oviducts, cervix uteri and vagina.
- Left testis, left epididymis and ovaries were fixed in BOUIN's solution.
- See table 7.8.1/1 for more details
Postmortem examinations (offspring):
SACRIFICE AND NECROPSY
- All surviving pups (after sacrifice on PND 4 with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Statistics:
- 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 and 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 and total sperm/g cauda epididymides: Pairwise comparison of the dose group with the control group using the Wilcoxon-test (one-sided) 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.
- Weight parameters after necropsy: Non-parametric one-way analysis using Kruskal-Wallis test (two-sided) was used. 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:
- Male mating index (%) = (number of males with confirmed mating / number of males placed with females) X 100
- Male fertility index (%) = (number of males proving their fertility / number of males placed with females) X 100
- Female mating index (%) = (number of females mated / number of females placed with males) X 100
- Female fertility index (%) = (number of females pregnant / number of females mated) X 100
- Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) X 100
- Live birth index (%) = (number of liveborn pups at birth / total number of pups born) X 100
- Post implantation loss (%) = [(number of implantations - number of pups delivered) / number of implantations] X 100
Offspring viability indices:
- Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on the day of birth) X 100
- Sex ratio = (number of live male or female pups on day of birth / number of live male and female pups on day of birth) X 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
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:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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

CLINICAL OBSERVATIONS FOR MALES AND FEMALES (EXCEPT GESTATION AND LACTATION PERIODS)
In male animals given 200 mg/kg bw/d, salivation after treatment (animal Nos. 33, 35, 37, 38, 39 and 40) was observed starting after 1 week of treatment. After 2 weeks of treatment, salivation before treatment was seen in male animals given 200 mg/kg bw/day (animal Nos. 31, 32, 33, 39 and 40). In addition, respiration sounds were observed predominantly after treatment in animal Nos. 33, 37 and 38, temporarily during the administration period. Eyelid halfclosure was also observed in 1 male (No. 37), although only in week 0. In females of given 200 mg/kg bw/d, salivation was observed most of the time after treatment in all animals during premating. Respiration sounds (predominantly after treatment) were observed in 5 animals, occasionally during premating. These findings were assessed as being related to treatment.
In test group treated at 75 mg/kg bw/d, one male animal (No. 24) 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.

In test group given 25 mg/kg bw/d, alopecia was observed in one female animal (No. 114) in the seventh week of treatment. This single finding was clearly incidental and not related to the test substance.

In test group given 200 mg/kg bw/d, 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 (Nos. 136 and 137) and piloerection (No. 136) were observed. These findings were assessed as being related to treatment. In test group given 75 mg/kg bw/d, respiration sounds after treatment were observed in one animal (No. 130) and only on gestation days (GD) 3 and 4. These two single occurrences were clearly without any toxicologically relevance and, therefore, assessed as being not related to treatment. One sperm positive female of test group 1 (No. 119 – 25 mg/kg bw/d), two sperm positive females of test group 2 (Nos. 125 and 128 – 75 mg/kg bw/d) and two sperm positive females of test group 3 (Nos. 136 and 138 – 200 mg/kg bw/d) did not deliver any F1 pups. Female rats No. 119 and No. 125, however, had implantation sites in utero indicating their fertility.

CLINICAL OBSERVATIONS FOR FEMALES DURING LACTATION
In test group given 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 (No. 137) and piloerection (No. 133) was observed in each one female animal temporarily during lactation.
These findings were assessed as being related to treatment.


BODY WEIGHT (PARENTAL ANIMALS)
- At 200 mg/kg bw/day, decreased body weight gain was noted in male animals in the first week of treatment (14.6 g vs. 27.5 g in control group).
- At dose levels of 75 and 200 mg/kg bw/day, decreased absolute terminal body weights were seen in males (96 and 95 %, respectively) before sacrifice. However a fasting period (withdrawal of food) was performed for at least 16-20 hours before autopsy.
- For details, refer figure 7.8.1/1

FOOD CONSUMPTION (PARENTAL ANIMALS)
- No treatment-related changes were noted.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- No significant treatment-related changes were noted.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Gestation index: The mean duration of gestation was significantly extended at 75 mg/kg bw/day (22.4 days) when compared to the control (21.9 days) whereas other test groups showed no significant differences. With regard to the fact that the mean duration of gestation at 75 mg/kg bw/day was only slightly outside the range of the historical control data (21.5-22.3 days) and no significant changes were noted in high-dose group the significant difference at 75 mg/kg bw/day was assessed as being spontaneous in nature and not test substance-related.
- Postimplantation loss (mean value): It was significantly increased at 75 mg/kg bw/day (1.3 vs. 0.1 in control group). Due to the fact that no increase was noted in high-dose group (200 mg/kg bw/day) 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 treated at 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 No. 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 (PART III, Supplement). However, a dose-response relationship was not observed with regard to test group given 200 mg/kg bw/d.
- No other significant treatment-related changes were noted.

SEX ORGAN WEIGHTS (PARENTAL ANIMALS)
- No significant treatment-related changes were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- No treatment-related changes were noted.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- No treatment-related changes were noted.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction/developmental toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment-related effect was observed at any dose level: clinical signs; mortality; body weight change, food consumption; gross pathology; organ weights and histopathology.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
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)

VIABILITY (OFFSPRING)
- At 200 mg/kg bw/day, viability index decreased significantly by 6 % (93 % at 200 mg/kg bw/day vs. 99 % in control group). A relation to treatment was assumed although gross necropsy revealed no relevant findings. However, it is worth noting that the loss of 6 of the 7 pups happened occurred on the first post natal day. In addition, two of the decedents pups came from a litter that started with an exceptional large amount of 17 pups (highest amount in the study), and four came from litter 133 that at birth was already weak with pup weights varying 10-40% below normal and many showing an underdeveloped appearance (Average pup weight about 25% below the overall average pup weight of the high dose group, and three pups that died on day 1 and were not weighed).
- No treatment-related changes were noted at dose-levels of 25 and 75 mg/kg bw/day.
- For details, refer figure 7.8.1/2

CLINICAL SIGNS (OFFSPRING)
- No significant treatment-related changes were noted.

BODY WEIGHT (OFFSPRING)
- No significant treatment-related changes were noted.

GROSS PATHOLOGY (OFFSPRING)
- No gross findings were observed during pup necropsy in any test group.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) for reproductive performance and fertility in male and female Wistar rats was considered to be 75 mg/kg bw/day. The NOAEL for general, systemic toxicity was 75 mg/kg bw/day for the F0 parental males (based on impaired body weight data) and 200 mg/kg bw/day for females. The NOAEL for developmental toxicity was 75 mg/kg bw/day for both sexes as the viability index was slightly decreased at a dose level of 200 mg/kg bw/day.
Executive summary:

In a reproduction / developmental toxicity screening test conducted according to the OECD Guideline 421 and in compliance with GLP, N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) was administered daily by oral gavage to groups of Wistar rats (10/sex/dose) at the dose-levels of 0, 25, 75 and 200 mg/kg bw/day. 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. Parental (F0) animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). 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 weekly 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 and examined macroscopically for external and visceral findings. All surviving F0 parental animals were sacrificed and assessed by gross pathology. Sex organ weights (testes, epididymides and ovaries) were recorded and a histopathological examination was performed when required.

In parental animals, no test substance-related mortalities occurred in any test group. Clinical signs including salivation; respiration sounds; eyelid-halfclosure and/or piloerection were observed on several days at 200 mg/kg bw/day. Salivation, noted at dose-levels of 75 and 200 mg/kg bw/day, was not considered to be an adverse and toxicologically relevant effect. At 200 mg/kg bw/day, decreased body weight gain was noted in male animals in the first week of treatment (53 %). At dose levels of 75 and 200 mg/kg bw/day, decreased absolute terminal body weights were seen in males (96 and 95 %, respectively) before sacrifice but after a fasting period of 16 -20 hours. No other treatment-related changes were noted at any dose-level in food consumption, reproductive performance, sex organ weights and no gross-finding was observed during gross necropsy.

In pups, viability index decreased significantly by 6 % at 200 mg/kg bw/day as mortality was observed essentially in two pups litters on the first postnatal day. A relation to treatment was assumed although gross necropsy revealed no relevant findings. No significant treatment-related changes were noted in clinical signs, body weight and at gross-necropsy.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of N-[3-(dimethylamino)propyl]-C18 unsaturated-alkylamide (unsaturated C18) for reproductive performance and fertility in male and female Wistar rats was considered to be 75 mg/kg bw/day. The NOAEL for general, systemic toxicity was 75 mg/kg bw/day for the F0 parental males (based on impaired body weight data) and 200 mg/kg bw/day for females. The NOAEL for developmental toxicity was 75 mg/kg bw/day for both sexes as the viability index was decreased at the dose level of 200 mg/kg bw/day.