Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A study was conducted to determine the potential adverse effects of the test substance when administered in the diet for at least 90 d and through one-generation of reproduction according to OECD Guidelines 408 and 422, in compliance with GLP.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 14, 2009 to December 15, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 408
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The animal model, the Crl:CD(SD) rat, is recognized as appropriate for subchronic toxicity and reproduction studies. In addition, WIL Research Laboratories, LLC has extensive historical control data for subchronic and reproductive studies conducted using Crl:CD(SD) rat.
Sex:
male/female
Details on test animals and environmental conditions:
Sexually mature male and virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC, were used as the test system on this study. Rats were approximately 6.5 weeks old at experimental initiation and were sexually mature at the time of mating.
Route of administration:
oral: feed
Details on exposure:
VEHICLE IDENTIFICATION
PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) was used in preparation of the control and test diets.

PREPARATION
Diets containing the test substance were prepared on a weight/weight basis in the following manner for all animals. An appropriate amount of the test substance into tared glass jars and transferred into a Hobart mixer with 2500 grams of rodent feed (weight/weight). The formulation was mixed for 3 minutes; the resultant formulation was termed pre-mix. The remainder of the rodent feed used to achieve the desired concentration was weighed and placed in the Hobart mixer. The diet was mixed for 10 minutes to achieve a total batch of homogeneous diet at the appropriate concentration per test group.
Concentration of the test diet was based on mean body weight and food consumption for that group from the previous week in an attempt to administer a constant dose on a mg/kg/day level. The female test diets were calculated using mean body weight and food consumption data for the toxicology phase females or reproductive phase females as appropriate.
Concentration of test diets during mating, gestation, and lactation were determined based on the mean food consumption and body weight data for the reproductive phase females from the last week prior to mating (whether or not mating was confirmed). During the breeding period, males were fed the lower concentration test diet to prevent overexposure.

EXPOSURE
The control and test diets were offered ad libitum to the males and reproductive phase females for a minimum of 70 consecutive days prior to mating. The males continued to receive the control and test diets throughout mating until euthanasia. The reproductive phase females continued to receive the control and test diets throughout mating, gestation, and lactation until euthanasia. The toxicology phase females were offered the control and test diets ad libitum for at least 90 days until euthanasia. The males and reproductive phase females were exposed for the total of 130 and 113-128 consecutive days, respectively, and the toxicology phase females were exposed for a total of 91 consecutive days.
The offspring of the reproductive phase females were potentially exposed to the test diet in utero as well as via milk while nursing and via direct diet consumption of maternal test diet during the later portion of the pre-weaning period. Dietary exposure levels were selected based on the results of previous studies.
Details on mating procedure:
The males and reproductive phase females were paired on a 1:1 basis within each treatment group following 70 days of exposure to the test diet. All animals were randomly selected for pairing, avoiding sibling mating. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. The males and reproductive phase females was mated once to produce one litter per generation (the F1 litters). Prior to pairing (Study Week 10), male body weights ranged from 404 g to 614 g and female body weights ranged from 190 g to 356 g. The animals were approximately 16 weeks old. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was determined on an 8 kg batch size at test diet concentrations of 50 and 13,000 ppm and room temperature stability for up to 10 days was determined on an 8 kg batch size at a test diet concentration of 13,000 ppm.
Samples for concentration analysis were collected weekly from the middle stratum of each formulation (including the control group). Concentration samples were analyzed for test substance concentration weekly for the first 4 weeks of the study and once a month thereafter for the remainder of the in-life phase.
Duration of treatment / exposure:
The test or basal diets were offered to all males for at least 70 days prior to mating and continued for at least 130 days until euthanasia. The appropriate diets were offered to the reproductive phase females for at least 70 days prior to mating and continued throughout mating, gestation, and lactation until euthanasia for a total of 113-128 days.
Details on study schedule:
28 April 2009.......................................... GLP experimental starting date (animal
receipt)
29 April 2009.......................................... Study initiation date (protocol signed by
Study Director)
14 May 2009........................................... Initiation of test diet exposure (experimental
start date)
14 May - 12 August 2009....................... Toxicology phase female test diet exposure
14 May - 18 September 2009 ................. Reproductive phase female test diet
exposure
14 May - 20 September 2009 ................. Male test diet exposure
22 July - 4 August 2009.......................... Mating period
23 July 2009............................................ First gestation day 0
13 August 2009....................................... Toxicology phase female necropsy
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five groups consisting of 12 males, 12 reproductive phase females, and 12 toxicology phase females
Control animals:
yes, concurrent no treatment
Details on study design:
The experimental design for this study consisted of a reproductive phase with four test-substance groups and one control group composed of 12 rats/sex each. In addition, a toxicology phase consisted of four test-substance groups and one control group composed of 12 females each. The selected animals were approximately 6 weeks old at the initiation of dietary substance exposure. Male body weight ranged from 160 g to 219 g, toxicology phase female body weights ranged from 145 g to 189 g, and reproductive phase female body weights ranged from 130 g to 172 g on the initial day of dietary substance exposure.
Parental animals: Observations and examinations:
All reproductive phase females were allowed to deliver naturally and rear their young to PND 21. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia (difficulty in delivery).
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrus of each reproductive phase female for 21 days prior to pairing and continuing until evidence of mating was observed. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation.
Litter observations:
On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

LITTER VIABILITY AND DEATHS

Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring dying were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Cannibalized pups were noted as such and discarded without further evaluation. Pups not expected to survive between birth and PND 4 to the next observation period (moribund) were euthanized via an intraperitoneal injection of sodium pentobarbital and necropsied. During PND 5 to weaning, moribund pups were euthanized by an intraperitoneal injection of sodium pentobarbital (prior to PND 11). A detailed gross necropsy was performed on any pup dying after PND 4; gross lesions were saved for possible future histopathological examination. The carcass of each pup was then discarded.

LITTER REDUCTION

To reduce variability among the litter, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. All selections were performed by computerized randomization. The remaining offspring were weighed, euthanized by intrathoracic injection of sodium pentobarbital and discarded on PND 4 without necropsy.

CLINICAL OBSERVATIONS

Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 10, 14, 17, and 21. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS

Pups were individually weighed on PND 1, 4, 7, 10, 14, 17, and 21. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.

SEX DETERMINATION

Pups were individually sexed on PND 0, 4, 14, and 21.
Postmortem examinations (parental animals):
A complete necropsy was conducted on all animals euthanized in extremis or at the scheduled termination. The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The number of former implantation sites was counted for all reproductive phase females, and the number of unaccounted sites was calculated by subtracting the number of pups born from the number of implantation sites. Tissue collection in reproductive phase females: cervix, uterusa, mammary gland vagina ovaries with oviducts, all gross lesions (when possible), pituitary.
Postmortem examinations (offspring):
All surviving F1 rats were euthanized by carbon dioxide inhalation and necropsied on PND 21. Organs and tissues were saved in 10% neutral-buffered formalin for possible future histopathological examination only as deemed necessary by the gross findings. Representative specimens with external malformations not pertaining to the skeletal system were preserved in 10% neutral-buffered formalin.
Statistics:
All statistical analyses were conducted using SAS version 9.1 (SAS Institute, Inc., 2002-2003) software. Locomotor activity data were analyzed by BioSTAT Consultants, Inc., Portage, MI.
Reproductive indices:
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of estrus of each reproductive phase female for 21 days prior to pairing and continuing until evidence of mating was observed.
Offspring viability indices:
Each litter was examined daily for survival, and all deaths were recorded.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg/day group: reproductive phase females and included red material around the nose observed predominantly during the gestation and/or lactation periods. Yellow material around the urogenital area was occasionally noted during exposure for the 600 mg/kg/day males and reproductive phase females.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg/day: lower mean body weights and body weight gains (or losses) and reduced food consumption and food efficiency in both sexes generally throughout the exposure period. These effects were more prevalent for the males and reproductive phase females with body weights that were up to 16.4% and 34.3% lower than concurrent controls, respectively, versus 7.2% lower in the toxicology phase females and without a corresponding effect on food consumption.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The higher reticulocyte count noted in the 600 mg/kg/day toxicology phase females correlated with the higher mean cell volume and was consistent with increased erythropoiesis. Lower absolute and percent eosinophil counts were also noted in the 600 mg/kg/day group males; however, because the mean absolute value was within the historical control data, these differences were not considered adverse.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Developmental effects at the 600 mg/kg/day exposure level were evident as a lower mean number of implantation sites resulting in lower mean number of pups born and live litter size.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on reproductive performance, locomotor activity (no remarkable shifts in the pattern of habituation), or FOB evaluations (home cage, handling, open field, neuromuscular, and physiological observations) noted at any exposure level.
Key result
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
other: reproductive
Organ:
ovary
pituitary gland
uterus
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Remarks on result:
not measured/tested
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weights in the 600 mg/kg/day group were slightly lower than the control group at birth and were approximately 56% lower than the concurrent control group on PND 21.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Conclusions:
Under the study conditions, there was no evidence of toxicity at exposure levels of 15, 50 and 175 mg/kg bw/day. Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive and developmental NOAEL was considered to be 175 mg/kg bw/day.
Executive summary:

A study was conducted to determine the potential adverse effects of the test substance when administered in the diet for at least 90 d and through one-generation of reproduction according to OECD Guidelines 408 and 422, in compliance with GLP. The study included evaluation of effects on male and female reproductive processes including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and on growth and development of the offspring through weaning. The males and reproductive phase females were bred to produce one litter. This study included five groups of Crl:CD(SD) rats consisting of 12 males, 12 reproductive phase females and 12 toxicology phase females. The treatment groups in each phase were offered a diet containing test substance at concentrations adjusted weekly to provide a target test substance consumption of 15, 50, 175 and 600 mg/kg bw/day. A concurrent control group received the basal diet on a comparable regimen. All animals were approximately 6 weeks of age at the initiation of diet exposure. The test or basal diets were offered to all males for at least 70 d prior to mating and continued for at least 130 d until euthanasia. The appropriate diets were offered to the reproductive phase females for at least 70 d prior to mating and continued throughout mating, gestation, and lactation until euthanasia for a total of 113-128 d. The toxicology phase females were euthanized after a maximum of 91 d of exposure to the appropriate diet. The summary of examinations and results of the clinical toxicity are included in repeated dose toxicity section. Vaginal lavages were performed daily for the reproductive phase females for determination of estrous cycles beginning 21 d prior to pairing. All reproductive phase females were allowed to deliver and rear their offspring to Lactation Day 21. F1 pups were necropsied on Postnatal Day (PND) 21. Each female in the reproductive and toxicology phases and all males received a complete detailed gross necropsy following the F1 pup necropsies or after at least 90 d of diet exposure; selected organs were weighed. Designated tissues from the males and toxicology phase females in the control and 600 mg/kg bw/day groups were examined microscopically. There was no test substance-related mortality or moribundity noted at any exposure level. However, test substance-related clinical findings were observed in the 600 mg/kg bw/day group reproductive phase females and included red material around the nose observed predominantly during the gestation and/or lactation periods. Yellow material around the urogenital area was occasionally noted during exposure for the 600 mg/kg/day males and reproductive phase females. Additionally, test substance-related lower absolute and relative-to-brain ovary, uterus, and pituitary weights were noted in the 600 mg/kg bw/day group reproductive phase females. There were no test substance-related effects on reproductive performance. Developmental effects at the 600 mg/kg bw/day exposure level were evident as a lower mean number of implantation sites resulting in lower mean number of pups born and live litter size. Additionally, mean pup body weights in the 600 mg/kg bw/day group were slightly lower than the control group at birth and were approximately 56% lower than the concurrent control group on PND 21. Postnatal survival was unaffected by test substance exposure and there were no test substance-related macroscopic findings noted for pups at necropsy. Under the study conditions, there was no evidence of toxicity at exposure levels of 15, 50 and 175 mg/kg bw/day. Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive and developmental NOAEL was considered to be 175 mg/kg bw/day (Edwards, 2010).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
175 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
There is an OECD 422 reproduction screening test, where the pre-mating dosing was extended to 70-days so it also incorporated the OECD408 requirements. This premating dosing ensured that the males were administered the test substance for the whole of the sperm production cycle. The study is therefore a valid assessment of the rats reproductive performance.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
N-[3-(dimethylamino)propyl] C6-9 alkyl amides mildly irritating to skin but severely irritating to eyes, so any local effects would be limited to irritation. It is of low volatility, so any inhalation would be due to exposure to aerosols, these would be unlikely to reach the lungs but could cause local irritation in the upper respiratory tract. The ECHA Guidance document allows the calculation of inhalation DNELs based on the oral NOAEL. The guidance assumes 50% oral and 100% inhalation absorption, so the oral NOAEL is adjusted for this prior to conversion to a NOEC. No testing is justified which avoid unnecessary use of additional animals.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
N-[3-(dimethylamino)propyl] C6-9 alkyl amides was mildly irritating to skin, it shows low acute dermal toxicity, with the LD 50 higher than for the oral route. The ECHA Guidance document allows the calculation of dermal long term DNELs based on the oral NOAEL. This is conservative approach as dermal absorption can be expected to be less than by the oral route. No testing is justified which avoid unnecessary use of additional animals.
Additional information

There were no test substance-related effects on reproductive performance. Developmental effects at the 600 mg/kg bw/day exposure level were evident as a lower mean number of implantation sites resulting in lower mean number of pups born and live litter size. Additionally, mean pup body weights in the 600 mg/kg bw/day group were slightly lower than the control group at birth and were approximately 56% lower than the concurrent control group on PND 21. Postnatal survival was unaffected by test substance exposure and there were no test substance-related macroscopic findings noted for pups at necropsy. Under the study conditions, there was no evidence of toxicity at exposure levels of 15, 50 and 175 mg/kg bw/day. Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive and developmental NOAEL was considered to be 175 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

There is a study was conducted to determine dose levels of the test substance for a possible definitive prenatal developmental toxicity study in rats according to OECD Guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The test substance in the vehicle, deionized water, was administered orally by gavage.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 02, 2009 to July 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)1
Details on test animals and environmental conditions:
SOURCE

Sexually mature, virgin female Crl:CD(SD) rats from Charles River Laboratories, Inc., Raleigh, NC, were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. Test facility has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.

ANIMAL RECEIPT AND ACCLIMATION

The animals were approximately 70 days old upon receipt. Each female was examined by a qualified technician on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number and housed for 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and general changes in appearance and behavior. Body weights were recorded prior to the initiation of breeding.

ANIMAL HOUSING

Upon arrival and until pairing, all rats were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. The cage-board was changed at least three times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required, as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

DIET, DRINKING WATER, AND MAINTENANCE

The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer. Feed lots used during the study are documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility is regularly sampled for contaminants. The results of the diet and water analyses are maintained at the test facility.

ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys® DDC Electronic Environmental control system. Actual mean daily temperature ranged from 70.2°F to 71.2°F (21.2°C to 21.8°C) and mean daily relative humidity ranged from 43.6% to 52.7% during the study. Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan), once daily during gestation days 6-19. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance formulations were weight/weight (test substance/vehicle) mixtures. The test substance formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots for daily dispensation, and stored at room temperature.
Prior to the initiation of dose administration, samples for homogeneity determination, resuspension homogeneity, and stability and concentration analysis were collected. All analyses were conducted using a validated high performance liquid chromatography method using ultraviolet absorbance detection.
The analyzed dosing formulations were within the standard operating procedures range for suspensions (85% to 115%), were homogeneous, and were stable after 10 days of room temperature storage.
Details on mating procedure:
At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding.
Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was prepared. The selected females were approximately 12 weeks old when paired for breeding. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.


Duration of treatment / exposure:
from gestation days (GD) 6 through 19
Frequency of treatment:
once daily
Duration of test:
90 d
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, in the vehicle, deionized water, was administered orally by gavage to five groups of eight bred female Crl:CD(SD)1 rats once daily from gestation days (GD) 6 through 19. Dose levels were 15, 50, 150, 500, and 1000 mg/kg/d administered at a dose volume of 10 mL/kg. A concurrent control group composed of eight bred females received the vehicle on a comparable regimen.

The bred females were assigned to groups using a WTDMS computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Replacement animals were arbitrarily assigned based on body weight. One female in the 1000 mg/kg/d group was found dead on gestation day 7. The cause of death for this female was determined to be an intubation error. Subsequently, this female was replaced with another female. Body weight values ranged from 233 g to 284 g on gestation day 0.
Maternal examinations:
CLINICAL OBSERVATIONS AND SURVIVAL

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20 (prior to dose administration during the treatment period). Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS

Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20. When body weights could not be determined for an animal during a given interval (due to an unscheduled death), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables. Gravid uterine weight was collected and net body weight (the gestation day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0 to 20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION

Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/d and g/kg/d for the corresponding body weight change intervals. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.
Ovaries and uterine content:
ANATOMIC PATHOLOGY AND LAPAROHYSTERECTOMY UNSCHEDULED DEATHS

A gross necropsy was performed on females that died or were euthanized (by carbon dioxide inhalation) in extremis during the course of the study. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The number and location of implantation sites, corpora lutea, and viable fetuses were recorded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). The maternal animals and all products of conception were then discarded.

GESTATION DAY 20 LAPAROHYSTERECTOMY

The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All surviving rats were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
Fetal examinations:
Fetal examinations were performed blind to treatment group. A detailed external examination of each fetus was conducted to include, but was not limited to, an examination of the eyes, palate, and external orifices. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). Each fetus was weighed, sexed, euthanized by hypothermia followed by an intrathoracic injection of sodium pentobarbital (if necessary), and discarded.
Statistics:
All statistical tests were performed using the WTDMS™ Management System. Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses.
Statistical analyses were not conducted if the number of animals was two or less. Due to the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ by ±1 in the last significant figure. Where applicable, the litter was used as the experimental unit.
Mean maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings noted for the females in the 1000 mg/kg/d group on the day prior to or day of death or euthanasia included prostration, hypoactivity, clonic convulsions, gasping, rales, shallow respiration, red and/or clear material around the nose and mouth, and dilated pupils; these findings were noted at the daily examinations or 1 hour following dose administration. Additionally, two females in the 500 mg/kg/d group were euthanized in extremis on gestation day 9 or 14. These females in the 500 mg/kg/d group were noted with increased respiration, decreased defecation, rales, gasping, labored respiration, and/or red and/or clear material around the nose, mouth, and/or forelimbs on the day of or day prior to euthanasia. All other animals survived to the scheduled necropsy.
Clinical findings observed for surviving females in the 500 mg/kg/d group at the daily examinations or approximately 1 hour following dose administration included rales, labored respiration, hair loss on the hindlimbs and/or thoracic and/or abdominal areas, clear material around the nose, mouth, eye and/or forelimbs, salivation, and/or dilated pupils.
Clinical findings noted in the 15, 50, and 150 mg/kg/d groups at the daily examinations or 1 hour following dose administration, including hair loss on various body surfaces, red and/or clear material around the mouth and/or nose, and/or yellow material on the urogenital area, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
All females in the 1000 mg/kg/d group were found dead or euthanized in extremis on gestation day 6, 7, or 8 (following 1-3 doses). Most females had body weight losses and reduced food consumption prior to death or euthanasia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant (p<0.01) mean body weight loss was noted in the 1000 mg/kg/d group at the onset of treatment (gestation day 6-7). Additionally, a body weight loss was noted for the one remaining female in the group on gestation day 7-8. Further evaluation of body weight data was precluded by death or euthanasia of all females in this group by gestation day 8.
In the 500 mg/kg/d group, a significant (p<0.05) mean body weight loss was noted during gestation days 6-9. Mean body weight gains in this group were significantly (p<0.01) lower during gestation days 15-20. When the overall treatment period (gestation day 6-20) was evaluated, mean body weight gain in the 500 mg/kg/d group was significantly (p<0.01) lower than the control group. The mean body weight in the 500 mg/kg/d group was significantly (p<0.05) lower (12.8%) than the control group on gestation day 20. Additionally, mean net body weight and net body weight gain in the 500 mg/kg/d group were lower (significant at p<0.01 for net body weight gain) than the control group. Mean gravid uterine weight in the 500 mg/kg/d group was lower than the control group primarily due to a single female with one implantation.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 15, 50, and 150 mg/kg/d groups were similar to that in the control group. The slight differences were not statistically significant and did not occur in a dose-related manner.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly (p<0.01) lower mean food consumption was noted in the 1000 mg/kg/d group at the onset of treatment (gestation day 6-7). Additionally, lower food consumption (6 g/animal/d) was noted for the one remaining female in the group on gestation day 7-8. Further evaluation of food consumption data in the 1000 mg/kg/d group was precluded by death or euthanasia of all females in this group by gestation day 8.
In the 500 mg/kg/d group, significantly (p<0.01 or p<0.05) lower mean food consumption was noted throughout the gestation treatment period (gestation days 6-9, 9-12, 12-15, 15-20, and 6-20). Lower mean food consumption noted in the 500 mg/kg/d group correlated to lower mean body weight gains during the treatment period.
Mean food consumption in the 15, 50, and 150 mg/kg/d groups was similar to that in the control group. The slight differences in food consumption were not statistically significant and did not occur in a dose-related manner.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All eight females in the 1000 mg/kg/d group were found dead or euthanized in extremis by gestation day 8. Additionally, two females in the 500 mg/kg/d group were euthanized in extremis on gestation day 9 or 14. All other females survived to the scheduled necropsy on gestation day 20. Macroscopic findings in both the 500 and 1000 mg/kg/d groups consisted of distended stomach and red material on the mouth and nasal area. In the 500 mg/kg/d group, one female had a distended stomach, duodenum, jejunum, ileum, cecum, and colon, and another female had thick vaginal contents. All females were gravid with the exception of one female each in the 50 and 1000 mg/kg/d groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A decrease in the mean number of viable fetuses and an increase in the mean litter proportion of pre-implantation loss was noted at 15 (12.9 per dam and 15.0% per litter, respectively) and 500 mg/kg/d (12.2 per dam and 21.5% per litter, respectively) compared to the concurrent control group (15.1 per dam and 7.1% per litter, respectively). This was due to one female in the 15 and 500 mg/kg/d groups that had one to three implantations. Because implantation occurs prior to the initiation of test substance administration, these differences were not attributed to test substance administration. Mean numbers of corpora lutea and implantation sites were similar across all groups. The slight differences from the control group were not statistically significant.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
Evaluation of laparohysterectomy parameters in the 1000 mg/kg/d group was precluded by mortality and moribundity. Intrauterine growth and survival were unaffected by test substance administration at dose levels of 15, 50, 150, and 500 mg/kg/d.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gains, and food consumption at dose levels of 500 and 1000 mg/kg/d when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg/d as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg/d.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
not measured/tested
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
no
Conclusions:
Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively.
Executive summary:

A study was conducted to determine dose levels of the test substance for a possible definitive prenatal developmental toxicity study in rats according to OECD Guideline 414 and EPA OPPTS 870.3700, in compliance with GLP. The test substance in the vehicle, deionized water, was administered orally by gavage to 5 groups of 8 bred female Crl:CD(SD)1 rats once daily from Gestation Days (GD) 6 through 19. Dose levels were 15, 50, 150, 500 and 1000 mg/kg bw/day administered at a dose volume of 10 mL/kg bw. A concurrent control group composed of 8 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity, and individual clinical observations were recorded from GD 0 to 20. Body weights and food consumption were recorded on GD 0 and 6-20 (daily). On GD 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external malformations and developmental variations. All eight females in the 1000 mg/kg bw/day group were found dead or euthanized in extremis on GD 6, 7 or 8. These females were noted with rales, hypoactivity, prostration, clonic convulsions, gasping, shallow respiration, red and/or clear material around nose and/or mouth, and dilated pupils at the daily examination or approximately 1 h following dose administration 1 d prior to or on the day of death or euthanasia. Additionally, body weight losses and reduced food consumption prior to death or euthanasia were noted for these females. The mortality at 1000 mg/kg bw/day precluded the evaluation of laprohysterectomy parameters and fetal morphology at this dose level. Two females in the 500 mg/kg bw/day group were euthanized in extremis on GD 9 or 14. Females in this group, including the two females that were euthanized in extremis, were noted with rales, increased respiration, dilated pupils, gasping, decreased defecation, labored respiration, salivation and/or clear and/or red material around the nose, mouth and/or forelimbs at the daily examinations or approximately 1 h following dose administration. All other females in the control, 15, 50 and 150 mg/kg bw/day groups survived to the scheduled necropsy on GD 20. There were no test-substance clinical findings noted at 15, 50 and 150 mg/kg bw/day at the daily examinations or approximately 1 h following dose administration. Lower mean body weight gain and decreased food consumption were noted at 500 mg/kg bw/day throughout the entire treatment period GD 6-20. As a result of lower mean body weight gains during gestation, mean body weights, net body weight gain and net body weight in this group were lower than the controls. A slightly lower mean gravid uterine weight was noted in the 500 mg/kg bw/day group due to a single female with one implantation. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 15, 50 and 150 mg/kg bw/day groups were similar to controls. At necropsy, two and one femalesat 500 and 1000 mg/kg bw/day, respectively, were noted with a distended stomach, and/or gas-filled distended stomach, duodenum, jejunum, ileum, cecum and colon. One female in the 500 mg/kg bw/day group (euthanized in extremis on GD 14) was noted with thick brown contents in the vagina. There were no internal findings noted at 15, 50 and 150 mg/kg bw/day. Intrauterine growth and survival at 15, 50, 150 and 500 mg/kg bw/day were similar to controls. There were no external malformations or developmental variations observed in this study. In conclusion, test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gain and food consumption at dose levels of 500 and 1000 mg/kg bw/day when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg bw/day as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg bw/day. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration at 15, 50, 150 and 500 mg/kg bw/day. Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively (Edwards, 2010).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
There is a good quality study which was done to determine dose levels of the test substance for a possible definitive prenatal developmental toxicity study in rats according to OECD Guideline 414 and EPA OPPTS 870.3700, in compliance with GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
N-[3-(dimethylamino)propyl] C6-9 alkyl amides mildly irritating to skin but severely irritating to eyes, so any inhalation would be limited to irritation. It is of low volatility, so any inhalation would be due to exposure to aerosols, these would be unlikely to reach the lungs but could cause local irritation in the upper respiratory tract. The ECHA Guidance document allows the calculation of inhalation DNELs based on the oral NOAEL. The guidance assumes 50% oral and 100% inhalation absorption, so the oral NOAEL is adjusted for this prior to conversion to a NOEC. No testing is justified which avoid unnecessary use of additional animals.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
N-[3-(dimethylamino)propyl] C6-9 alkyl amides was mildly irritating to skin, it shows low acute dermal toxicity, with the LD 50 higher than for the oral route. The ECHA Guidance document allows the calculation of dermal long term DNELs based on the oral NOAEL. This is conservative approach as dermal absorption can be expected to be less than by the oral route. No testing is justified which avoid unnecessary use of additional animals.
Additional information

Mortality at 1000 mg/kg bw/day, rats were terminated in extremis on days 6 -9, precluded the evaluation of laprohysterectomy parameters and fetal morphology at this dose level. Two females in the 500 mg/kg bw/day group were euthanized in extremis on GD 9 or 14. Females in this group, including the two females that were euthanized in extremis, were noted with rales, increased respiration, dilated pupils, gasping, decreased defecation, labored respiration, salivation and/or clear and/or red material around the nose, mouth and/or forelimbs at the daily examinations or approximately 1 h following dose administration. All other females in the control, 15, 50 and 150 mg/kg bw/day groups survived to the scheduled necropsy on GD 20. There were no test-substance clinical findings noted at 15, 50 and 150 mg/kg bw/day at the daily examinations or approximately 1 h following dose administration. Lower mean body weight gain and decreased food consumption were noted at 500 mg/kg bw/day throughout the entire treatment period GD 6-20. As a result of lower mean body weight gains during gestation, mean body weights, net body weight gain and net body weight in this group were lower than the controls. A slightly lower mean gravid uterine weight was noted in the 500 mg/kg bw/day group due to a single female with one implantation. Mean maternal body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights, and food consumption in the 15, 50 and 150 mg/kg bw/day groups were similar to controls. At necropsy, two and one femalesat 500 and 1000 mg/kg bw/day, respectively, were noted with a distended stomach, and/or gas-filled distended stomach, duodenum, jejunum, ileum, cecum and colon. One female in the 500 mg/kg bw/day group (euthanized in extremis on GD 14) was noted with thick brown contents in the vagina. There were no internal findings noted at 15, 50 and 150 mg/kg bw/day. Intrauterine growth and survival at 15, 50, 150 and 500 mg/kg bw/day were similar to controls. There were no external malformations or developmental variations observed in this study. In conclusion, test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gain and food consumption at dose levels of 500 and 1000 mg/kg bw/day when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg bw/day as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg bw/day. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration at 15, 50, 150 and 500 mg/kg bw/day. Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively.

Mode of Action Analysis / Human Relevance Framework

There were no adverse effects on reproduction even at clearly maternally toxic dose levels. The only effects on development were related to lower litter weights and increased post implantation loss seen in the presence of significant maternal toxicity. In the Developmental toxicity study there were no indications of structural abnormalities even in the presence of significant maternal toxicity. This supports the argument that the effects seen were a combination of direct toxicity on the foetus and secondary to toxicity to the mother which resulted in reduced food consumption which may have contributed to the reduced pup weight and higher post-implantation loss. General toxic effects might be expected to be relevant to human should very high exposure occur but are unlikely to occur at likely low human exposure levels.

Justification for classification or non-classification

N-[3-(dimethylamino)propyl] C6-9 alkyl amides has been tested in a modified OECD422 study, where the parental rats were dose for 70 days prior to mating. This ensured that the males were dose through the entire sperm cycle, maximising the possibility to detect any effect of dosing on male fertility. There were clearly no effects on male fertility or on female reproductive performance.

Systemic toxicity was exhibited at 600 mg/kg bw/day by clinical findings, lower mean body weights, body weight gains and food consumption for both sexes, and lower absolute and relative-to-brain ovary, uterus and pituitary weights for the reproductive phase females. In addition, lower mean live litter size on PND 0, number of pups born and implantation sites and lower mean pup weights were noted in the 600 mg/kg bw/day group in the presence of excessive maternal toxicity. At 175 mg/kg there were no effects on development in the absence of marked maternal toxicity.

In the preliminary study for the possible OECD414 study,test substance-related toxicity was evidenced by reductions in mean maternal body weights, body weight gain and food consumption at dose levels of 500 and 1000 mg/kg bw/day when test substance was administered orally by gavage to bred Crl:CD(SD) rats. In addition, the maximum tolerated dose was exceeded at 500 and 1000 mg/kg bw/day as evidenced by mortality and moribundity. There was no evidence of maternal toxicity at 15, 50, and 150 mg/kg bw/day. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration at 15, 50, 150 and 500 mg/kg bw/day. Under the study conditions, the maternal and developmental NOAEL of the test substance were 150 and 500 mg/kg bw/day, respectively.

Based on these results there is no evidence of specific toxicity to reproduction or development and therefore no requirement for classification for Toxicity to reproduction by EU CLP (GHS) criteria