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Administrative data

Description of key information

There are two high quality Klimisch 1 repeat oral dose studies in rats available. There is an OECD407 28-day study via the diet and an OECD422 study where the extended pre-mating administration of the test substance in the diet ensures that the duration of the study is at least 90 days and so provides the equivalent data as a 90-day feeding study. This study included 12 additional females in the controls and each of the four-treatment group which were not mated to ensure he full requirements for OECD 408 were complied with.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 09, 2009 to April 06, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species and strain of animal is recognized as appropriate for short-term toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant control data are available. The animals were approximately 7 weeks old at the initiation of dose administration.
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION/PRETEST PERIOD
37 male and 37 female Crl:CD(SD) rats were received in good health on 24 February 2009, from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 38 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed 3 days later. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) in the scapular area. All animals were housed for a 13-day acclimation/pretest period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Individual body weights and food consumption were recorded and detailed physical examinations were performed periodically during the pretest period.

ANIMAL HOUSING
Upon arrival, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Racks were rotated within the animal room at least once every 2 weeks during the study to change the physical location from one area of the room to another to ensure similarly varied environmental exposure for all animals. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Nylabones® were provided to all animals throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
The basal diet fed ad libitum during the acclimation period and to the control group throughout the study was PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal). The certified feed was analyzed by the manufacturer and results were provided to WIL Research Laboratories, LLC. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, the test diet, and the basal diet were provided ad libitum throughout the study, except during designated periods of fasting when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to the standard operating procedures. The results of the diet and water analyses are maintained at the test facility.

ENVIRONMENTAL CONDITIONS
All animals 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 ± 5 °F (22 ± 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 69.7 °F to 72.2°F (20.9 °C to 22.3 °C) and mean daily relative humidity ranged from 42.5% to 56.1% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
Four days prior to the initiation of dose administration, all available animals were weighed and examined in detail for physical abnormalities. Based on the review of all appropriate pretest data, animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure. A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification in a block design. The animals were then arranged into groups. Individual body weights at randomization were within ±20% of the mean for each sex. Groups 1-6 each consisted of 5 males and 5 females. Individual body weights ranged from 179 g to 212 g for males and from 150 g to 178 g for females at randomization.
Route of administration:
oral: feed
Details on route of administration:
The basal (control) diet or test diets were fed ad libitum for 28 consecutive days, through the day prior to the scheduled necropsy. Initial concentrations were based on average food consumption and body weights determined during pretest period. Concentration was adjusted weekly, based on the expected average weight and food consumption.
Vehicle:
other: meal
Details on oral exposure:
PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) was used to prepare the control and test diets. Each lot used was documented.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The control and test diets were prepared approximately weekly, placed in labeled storage bags and stored at room temperature. Dietary concentrations for the test diets were adjusted weekly based on the expected average weight and food consumption.
Samples for homogeneity determination were collected from the top, middle, and bottom strata of the 50 and 15,000 ppm pretest formulations.
Samples for stability determinations were collected from the middle strata of these same pretest formulations and from the basal diet. Samples for stability determination were analyzed following room temperature storage for 0, 5, 8, and 10 days. Samples for concentration analysis were collected from the first and last formulation preparation from the middle stratum of each dietary formulation (including the control group). All samples were stored frozen (approximately -65° to -85°C) until shipment for analysis.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basal (control) diet
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Dosage level changed from 10 mg/kg/day to 800 mg/kg/day on study day 18
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, was offered on a continuous basis in basal diet for 28 consecutive days to five groups (Groups 2-6) of Sprague Dawley (Crl:CD®[SD]) rats at dosage levels of 10, 50, 100, 300, and 1000 mg/kg/day, respectively. A concurrent control group (Group 1) received the basal diet on a comparable regimen. On study day 18, the dosage level for Group 2 was changed from 10 mg/kg/day to 800 mg/kg/day for the remainder of the study. All groups (Groups 1-6) consisted of five animals/sex. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded approximately weekly. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals at the scheduled necropsy (study week 4). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Tissues were retained for possible future microscopic examination.
Observations and examinations performed and frequency:
SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
Clinical examinations were performed once daily for all animals. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals beginning during the acclimation period, upon individual housing of the animals, on the day of randomization, and approximately weekly thereafter.

BODY WEIGHTS
Individual body weights were recorded approximately weekly, beginning at least one week prior to test substance administration and on the day prior to the scheduled necropsy. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded for all animals on the day of the scheduled necropsy.

FOOD AND TEST SUBSTANCE CONSUMPTION
Individual food consumption was recorded at least weekly during the pretest period and throughout the study. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals.
The mean amounts of test substance consumed (mg/kg/day) by each sex per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the food (mg/kg). Food efficiency (body weight as a percent of feed consumed) was also calculated for all animals.
Sacrifice and pathology:
CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected from all animals prior to the scheduled necropsy (study day 28). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for hematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurane. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium EDTA (hematology), sodium citrate (coagulation) or no anticoagulant (serum chemistry).

HEMATOLOGY AND COAGULATION
All red blood cells, white blood cells, plate indices and coagulation related parameters were sampled for analysis. Additionally serum chemistry and urinalysis were conducted.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Lungs, Ovaries with oviducts, Pituitary, Prostate, Spleen, Thymus, Thyroid with parathyroids.

MACROSCOPIC EXAMINATION
A complete necropsy was conducted for all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
Statistics:
All statistical tests were performed using the Toxicology Data Management System (WTDMS™). Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Mann-Whitney U-test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight loss and/or lower body weight gains were noted for the 300, 800, and 1000 mg/kg/day group males and females throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption were noted for the 300, 800, and 1000 mg/kg/day group males and females throughout the study. Clinical observations of decreased defecation in the 800 mg/kg/day group females correlated to lower food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The higher red blood cell counts, hemoglobin, hematocrit, and MCV levels and the lower reticulocyte counts in the 800 mg/kg/day group animals may be related to dehydration or malnutrition, correlating to the lower food consumption noted in this group. The opposite effect on reticulocyte counts and red cell mass in the 1000 mg/kg/day group animals may be due to an over compensation for an initial reduction in red blood cell counts.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lower glucose levels in the 800 mg/kg/day group animals and 1000 mg/kg/day group males in the serum may have resulted from inadequate food consumption and/or depletion of glycogen stores (Levin, 1993).
Lower values for liver enzymes alkaline phosphatase (800 mg/kg/day and 1000 mg/kg/day group females) and AST (all test substance-treated group females), in the absence of correlating gross organ observations, were likely due to reduced metabolic liver function corresponding to decreased food consumption.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Higher urea nitrogen in the 800 and 1000 mg/kg/day group animals and increased albumin and total protein in the 800 and 1000 mg/kg/day group males were most likely due to dehydration and malnutrition from lower food consumption.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related differences in organ weights consisted of lower liver, spleen, and thymus weights (absolute and relative to body and/or brain) in the 800 mg/kg/day group females. Liver enzymes were also altered and lower spleen and thymus weights may be due to stress. Direct test-substance effects cannot be confirmed without histopathologic examination.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, systemic toxicity of the test substance offered in the diet to rats for 28 d was observed at 800 (beginning on Day 18) and 1000 mg/kg bw/day, as evidenced by lower body weight gains, lower food consumption, as well as effects on hematology, serum chemistry parameters and organ weights. Therefore, the 28 d NOAEL, with no microscopic examination of tissues, was 300 mg/kg bw/day for males and females.
Executive summary:

A study was conducted to determine the potential toxicity of the test substance when administered to rats via the diet for 28 consecutive days and to assist in dose selection for the 90 d dietary definitive reproductive toxicity study according to OECD Guideline 407, in compliance with GLP. The test substance was offered on a continuous basis in basal diet for 28 consecutive days to 5 groups (Groups 2-6) of Sprague Dawley (Crl:CD (SD) rats at dosage levels of 10, 50, 100, 300 and 1000 mg/kg bw/day, respectively. A concurrent control group (Group 1) received the basal diet on a comparable regimen. On Day 18, the dosage level for Group 2 was changed from 10 to 800 mg/kg bw/day for the remainder of the study. All groups (Groups 1-6) consisted of 5 animals/sex. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded approximately weekly. Clinical pathology parameters (hematology, coagulation, serum chemistry and urinalysis) were analyzed for all animals at the scheduled necropsy (Week 4). Complete necropsies were conducted on all animals and selected organs were weighed at the scheduled necropsy. Tissues were retained for possible future microscopic examination. There were no test substance-related effects on survival, urinalysis parameters or macroscopic findings. However, lower food consumption was noted for the 800 and 1000 mg/kg bw/day males and females as compared to controls. The following effects were considered secondary to test substance-related effects of dehydration and lower food consumption: decreased defecation at 800 mg/kg/day; lower body weights and lower body weight gains at 300 (females only), 800 and 1000 mg/kg/day; higher red blood cell counts, hemoglobin, hematocrit and lower reticulocyte counts at 800 mg/kg bw/day and MCV levels in the 800 mg/kg bw/day group females; lower red blood cell counts, hemoglobin, hematocrit in the 1000 mg/kg bw/day females, and higher reticulocyte count levels in the 1000 mg/kg bw/day group animals; lower eosinophils at 800 and 1000 mg/kg bw/day; higher albumin and total protein at 800 and 1000 mg/kg bw/day, and increased urea nitrogen for 800 and 1000 mg/kg bw/day group animals. Additional possible test substance effects included lower glucose at 800 mg/kg (males and females) and 1000 (males) mg/kg bw/day, slightly lower values for liver enzymes at 800 and 1000 mg/kg bw/day; and lower liver, spleen and thymus weights (absolute and relative to body and/or brain) were noted in the 800 mg/kg/day group females. Under the study conditions, systemic toxicity of the test substance offered in the diet to rats for 28 d was observed at 800 (beginning on Day 18) and 1000 mg/kg bw/day, as evidenced by lower body weight gains, lower food consumption, as well as effects on hematology, serum chemistry parameters and organ weights. Therefore, the 28 d NOAEL, with no microscopic examination of tissues, was 300 mg/kg bw/day for males and females (Matthew, 2010).

Endpoint:
sub-chronic toxicity: oral
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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
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, test fecility has extensive historical control data for subchronic and reproductive studies conducted using Crl:CD(SD) rat.
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION/PRETEST PERIOD

Seventy male and 70 female Crl:CD(SD) rats (approximately 29 days of age) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 28 April 2009. An additional 74 females Crl:CD(SD) rats (approximately 36 days of age) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 5 May 2009. Animals were housed two to three per cage (by sex) for approximately 2 to 3 days to allow for adaptation to the automatic watering system. The day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals received on 28 April 2009 and 5 May 2009 were housed for acclimation periods of 16 and 9 days prior to the first day of treatment, respectively. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior. The animals were allowed a pre-treatment week during which detailed physical examinations were performed, body weight, and food consumption were recorded and general health was monitored, but animals did not receive the test diets.

ANIMAL HOUSING

Following the initial acclimation period and until pairing (for the reproductive phase animals), all animals were housed individually in clean, stainless steel wire-mesh cages suspended above the cage-board. The cage-board was changed at least three times per week. All animals were offered Nylabones® for the duration of the study except during the cohabitation period for the males and reproductive phase females. The males and reproductive phase females were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the reproductive phase females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons). The dams and their litters were housed in these cages until euthanasia on lactation day 21. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until euthanasia. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

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 a. Feed lots used during the study are documented in the study records. The test or control diet was provided ad libitum throughout the study except during the overnight period of fasting prior to blood collection when food, but not water, was withheld. Feeders were changed and sanitized once per week. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system was provided ad libitum throughout the study. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are maintained at test fecility. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives or interpretation of the results of this study.

ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room(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 20.8°C to 22.1°C and mean daily relative humidity ranged from 39.1% to 54.3% during the study. Light timers were calibrated to provide a 12-hour light (600 hours to 1800 hours)/12-hour dark photoperiod. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.



Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral 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.
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.
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.

BREEDING PROCEDURES

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.
Observations and examinations performed and frequency:
All animals were observed twice daily for mortality and moribundity. Clinical observations were performed once daily during the dietary exposure period. Detailed physical examinations were not performed on the day of Functional Observational Battery (FOB) assessments for the males and toxicology phase females.

Detailed physical examinations, body weights, and food consumption were recorded at least weekly for the males, toxicology phase females, and during the pre-mating period for the reproductive phase females. The reproductive phase female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 14, and 21. Food consumption was measured on gestation days 0-20 (daily) and lactation days 1-21 (daily) for the reproductive phase females. Food consumption was not recorded during the breeding period for the males and reproductive phase females.

Functional observational battery (FOB) observations were recorded for all males and all toxicology phase females during the last week of test substance administration (study days 123-126 for males and study days 85-89 for females). The FOB used at the test fecility is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1991a, Moser et al., 1991b, and O’Donoghue, 1989).

Locomotor activity counts were recorded for all males and toxicology phase females during the last week of exposure following approximately 125 days of dose exposure for the males and beginning approximately 86 days of dose exposure for the toxicology phase females. Locomotor activity, recorded after the completion of the FOB, was measured automatically using the Kinder Scientific MotorMonitor System (Kinder Scientific, LLC, Poway, CA).

Ocular examinations were conducted on males and toxicology phase females prior to randomization and during the final week of test diet administration. All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

CLINICAL PATHOLOGY AND URINALYSIS (MALES AND TOXICOLOGY PHASE FEMALES)

Blood samples for clinical pathology and urinalysis evaluations were collected from all males and toxicology phase females just prior to the scheduled necropsies. The animals were food-fasted overnight prior to blood collection with water available using metabolism cage in which urine was collected before taking the blood samples. Blood samples for serum chemistry and hematology were collected from the retro-orbital sinus following isoflurane anesthesia. Blood samples for coagulation parameters were collected from the vena cava at the time of euthanasia. Blood was collected into tubes containing potassium EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).

Vaginal lavages were performed daily for the reproductive phase females for determination of estrous cycles beginning 21 days 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.
Sacrifice and pathology:
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 days of diet exposure; selected organs were weighed. Designated tissues from the males and toxicology phase females in the control and 600 mg/kg/day groups were examined microscopically.
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.
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.
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 specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower absolute and relative-to-brain ovary, uterus, and pituitary weights were noted in the 600 mg/kg/day group reproductive phase females.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
organ weights and organ / body weight ratios
Remarks on result:
other: organ weights for the reproductive phase females
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
ovary
pituitary gland
uterus
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes
Conclusions:
Under the study conditions, there was no evidence of toxicity noted at exposure levels of 15, 50, and 175 mg/kg/d. Systemic toxicity was exhibited at 600 mg/kg/d 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/d group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive, and developmental NOAEL was considered to be 175 mg/kg/d.
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 days and through one-generation of a reproduction according to OECD Guideline 408 and 422, in compliance with GLP. The study included evaluation of the potential effects of the test substance 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 test substance 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/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 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. The toxicology phase females were euthanized after a maximum of 91 days of exposure to the appropriate diet. All animals were observed twice daily for mortality and moribundity. Clinical observations were performed once daily. Detailed physical examinations, body weights, and food consumption were recorded at least weekly for the males, toxicology phase females, and during the pre-mating period for the reproductive phase females. The reproductive phase female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1, 4, 7, 14, and 21. Food consumption was measured on gestation days 0-20 (daily) and lactation days 1-21 (daily) for the reproductive phase females. Food consumption was not recorded during the breeding period for the males and reproductive phase females. Functional observational battery (FOB) and locomotor activity were recorded for males and toxicology phase females during the last week of test diet exposure. Hematology, serum chemistry, and urinalysis parameters were evaluated for the males and toxicology phase females on the day of euthanasia. Ophthalmic examinations were performed on males and toxicology phase females prior to the initiation of test diet exposure and during the final week of test diet exposure. 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 days of diet exposure; selected organs were weighed. Designated tissues from the males and toxicology phase females in the control and 600 mg/kg/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/d 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. In addition, toxicity at the 600 mg/kg/day exposure level was further evidenced by 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. Additionally, test substance-related lower absolute and relative-to-brain ovary, uterus, and pituitary weights were noted in the 600 mg/kg/day group reproductive phase females. There were no test substance-related clinical findings or effects on body weight, food consumption, or organ weights noted at 15, 50, and 175 mg/kg/day. 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. There were no test substance-related ophthalmic, macroscopic or microscopic findings, serum chemistry, or urinalysis evaluations noted at any exposure level. However, 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/d group males; however, because the mean absolute value was within the historical control data, these differences were not considered adverse. 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. Under the study conditions, there was no evidence of toxicity noted at exposure levels of 15, 50, and 175 mg/kg/d. Systemic toxicity was exhibited at 600 mg/kg/d 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/d group in the presence of excessive maternal toxicity. Therefore, the systemic, reproductive, and developmental NOAEL was considered to be 175 mg/kg/d (Edwards, 2010).

Endpoint conclusion
Endpoint conclusion:
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 a Klimisch 1 28day study which was followed by a 90-day study incorporated in an OECD422 study including groups of none pregnant females. This provides high quality repeat dose toxicology data which is of Klimisch 1 quality fully GLP and guideline compliant. This provide a 90-day NOAEL which is a valid basis for the calculation of DNELs
System:
other: reduced bodyweight gain and food consumption

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Testing by inhalation is not scientifically justified as ECHA guidance allows the calculation of inhalation DNELs based on the available oral NOAEL data. This avoids the unnecessary use of additional rats.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
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 so no data on local inhalation effects are required.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Testing by the dermal route is not scientifically justified as ECHA guidance allows the calculation of Dermal DNELs based on the available oral NOAEL data. This avoids the unnecessary use of additional rats.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
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 so no data on local dermal effects are required.

Mode of Action Analysis / Human Relevance Framework

There are no specific adverse effects seen from repeated dietary administration only some mild general toxic effects at the 600 mg/kg top dose as shown by reduced bodyweight gain and food consumption. No mode of action can be determined form these results. It is unlikely that there would an specific target organ for toxicity in humans.

Additional information

Justification for classification or non-classification

At the 600mg/kg top dose in the 90day dietary study there was only mild none specific toxicity and certainly no specific target organ toxicity was seen. Therefore, there is no indication of effects that could require a STOT RE classification based on EU CLP (GHS) criteria.