4-[3-(1-naphthylamino)propyl]morpholine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650 (2000)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test Material Name: 4-(3-(1-naphthylamino)propyl)morpholine
Chemical Name: N-1-Naphthalenyl-4-morpholinepropanamine
Lot/Reference/Batch Number: ZA01212016
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 94.4 % by liquid chromatography with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry (Ferrer, 2016a).
Test Material Stability Under Storage Conditions: 4-(3-(1-Naphthylamino)propyl)morpholine, lot ZA01212016, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313 (Ferrer, 2016b).

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD) were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: Crl:CD(SD) were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River Laboratories (Raleigh, North Carolina)
Age at Study Start: Approximately 12 weeks
Health Status and Acclimation: Upon arrival all animals were acclimated to the laboratory for approximately one week prior to the study. During the acclimation period, each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. From the time of arrival through acclimation all animals were maintained on the TERC Institutional Animal Care and Use Committee (IACUC) approved Animal Housing for Acclimation protocol under the direction of the Attending Veterinarian until transferred to this protocol for study purposes. The Toxicology and Environmental Research and Consulting Laboratory was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).

Housing:
Upon arrival animals were housed two-three per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages containing corn cob bedding for the prebreeding portion of the study. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors in order to better visualize copulatory plugs. After breeding, males were returned to solid bottom stainless steel cages. During gestation and littering, dams (and their litters), and non-mated females were housed in plastic cages containing irradiated corn cob bedding from approximately GD 0 until LD 13. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
The following environmental conditions were targeted in the animal room from the day of arrival till necropsy, however on September 7, 2016; the humidity was 73%, which is about the targeted humidity. All animals appeared normal during the cage-side observations throughout the course of the study, indicating there was no negative impact on the study due to these excursions in environmental conditions. All observed ranges were documented in the study file.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Note: Photoperiod times may have changed due to study-related activities.

Feed and Water:
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses were maintained in the study file.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Route, Method of Administration, Frequency, Duration and Justification:
Oral gavage was the preferred route of exposure according to the relevant test guideline. Male rats were dosed daily for four weeks prior to mating and continuing throughout the mating for 58 days. Female rats were dosed once daily for four weeks prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (thirteen days).

Dose Levels and Justification:
The high-dose level of 200 mg/kg/day was selected based upon data obtained from a preliminary range-finding study and was expected to induce some effects on body weight gains and feed consumption during the first few days of dosing, but not cause death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity observed among the high-dose group rats and to establish a NOEL.

Dose Preparation:
The test material was administered in a propylene glycol (PG) vehicle, such that a dose volume of 6 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose suspensions were prepared periodically throughout the study period based upon stability. Dose suspensions were not adjusted for purity.

Solubility:
4-(3-(1-naphthylamino)propyl)morpholine was determined to be soluble in the propylene glycol at a concentration of 0.25 to 250 mg/ml (Fiting, 2016).

Details on mating procedure:

Breeding Procedure:
Breeding of the adults commenced after approximately four weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
Dose Confirmation and Homogeneity:
Analyses to determine concentration of the test material of all dosing suspensions from the first mix were initiated prior to the start of dosing. The low- and high-dose suspensions from the first mix of the main study were analyzed to confirm homogenous distribution of the test material concurrent with dose confirmation. Analyses were conducted using high performance liquid chromatography with ultraviolet detection (HPLC/UV) (Fiting, 2016).

Stability:
4-(3-(1-naphthylamino)propyl)morpholine has been shown to be stable in propylene glycol for at least 21 days at 0.25 - 250 mg/mL (Fiting, 2016).

Retainer Samples:
A sample of the solid test material was retained, but samples of the dose suspensions were not retained.

Duration of treatment / exposure:

Female rats were dosed once daily for approximately four weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through postpartum day 13. Male rats were dosed approximately four weeks prior to breeding and continuing through breeding (two weeks) for a minimum exposure period of 58 days.

Frequency of treatment:

Daily, by gavage.

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12/sex/dose group

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Daily In-Life Observations:
A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed for included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Cage-side examinations were conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Clinical Observations:
Clinical examinations were conducted on all animals pre-exposure and at least once daily throughout the study. During exposure period, these examinations were conducted approximately one hour after dosing. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings. Females were observed for signs of parturition beginning on or about GD 20 (see litter data).

Detailed Clinical Observations:
Detailed clinical observations (DCO) were conducted on all males pre-exposure and weekly throughout the study. Detailed clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated females received DCO examinations on GD 0, 7, 14, and 20. Females that delivered litters were subsequently evaluated on LD 7 and 13. Detailed clinical observations were not conducted on females that failed to mate or deliver a litter during the gestation and lactation phases of the study. The DCO was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).

Functional Tests:
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 13. Females that failed to deliver did not undergo functional testing during the last week of treatment.

Body Weights/Body Weight Gains:
All rats were weighed at least once during the pre-exposure period and twice during the first week of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1, 4, 7, and 13. Females that failed to mate or deliver a litter were weighed at least weekly for the remainder of the study. Body weight analyses were conducted for the following days: GD 0, 7, 14, and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4, 4-7, 7-13 and 1-13.

Feed Consumption:
The amount of feed consumed was determined twice during the first week of dosing, and then weekly during the four week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. During breeding, feed consumption was not measured for males or females due to co-housing. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1, 4, 7, 11, and 13. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)

Oestrous cyclicity (parental animals):

Estrous Cycle Evaluation:
Vaginal lavage samples from all females were collected daily for approximately two weeks pre-exposure, daily for two weeks immediately prior to mating, and during cohabitation until each female was sperm- or plug-positive or until the two week mating period elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring lavage fluid to a microscope slide. These vaginal lavage slides were examined microscopically and stage of estrous was recorded. The information was evaluated to determine estrous cycle length and pattern. On the day of scheduled necropsy, the stage of the estrous cycle was also determined for all female rats. The pre-exposure estrous data were documented in the study file.

Litter observations:

Litter Data:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, 4, 7, and 13, and the sex and body weight of each pup on LD 1, 4, 7, and 13. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily Observations). In addition, pup clinical observations were recorded on days 0, 1, 4, 7, and 13 postpartum. Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then discarded.

Anogenital Distance:
Anogenital distance (AGD; absolute and relative to the cube root of PND 1 body weight) was measured in all pups on PND 1 (Gallavan et al., 1999). The sequence of the data collection was counterbalanced across groups (e.g., control, high, middle, low) to the extent possible on each day to control for potential confounding influences of collection timing.

Culling and Culled Pups:
To minimize variation in pup growth due to differences in litter size, all litters were standardized to eight pups per litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated randomization procedure, so that four males and four females remained in each litter. If it was not possible to have four pups/sex in each litter, unequal numbers of males and females were retained (e.g., five males, three females). Culled pups were used for collection of contingency serum samples for thyroid hormone analysis and thyroids of these pups were saved (see below in Necropsy section). Litters with
≤ 8 pups were not culled. Preferential culling of runts was not performed.

Nipple/Areolae Retention:
All offspring were evaluated for the presence of nipple/areolae on PND 12 in accordance with the methods described by McIntyre et al. (2001). The average number of nipples/areolae in male and female offspring in each litter was determined. The grand mean number of nipples/areolae for males and females in each dose level was calculated from these litter means. Observers were blind to treatment group when evaluating pups for the presence of nipples/areolae.

Postmortem examinations (parental animals):

Clinical Pathology:
Adult animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter. Hematology, coagulation, clinical chemistry were evaluated. Urinalysis was also conducted. Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.

Thyroid Hormone Measurements:
Serum for Thyroid Hormone Analyses:
The sequence of the sample collection for all of the animals was counterbalanced across groups (e.g., control, high, middle, low) to the extent possible on each day of sample collection to control for potential confounding influences of collection timing. Blood was placed in serum separator tubes and placed on ice. Serum was separated from cells and stored in the freezer until analysis.
Serum T3 and T4 concentrations were assessed for all samples from adult males, LD 14 dams, PND 4 culled pups, and PND 13 pups. TSH was not assessed for any of the animals.

Anatomic Pathology:
Adult Necropsy:
Adult males (fasted) were submitted for necropsy after at least six weeks of exposure. Adult females (fasted) were terminated on lactation day 14, or 24-26 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, fasted rats were weighed in the animal room and submitted alive for necropsy. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen. While under anesthesia, blood was collected from the orbital sinus (all males, all females that littered). The animals were then placed in a CO2 chamber to continue anesthesia. Under a deep plane of anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al., 1964 and examined for the presence and number of implantation sites. Uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues listed in Table 4 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues.

Histopathology:
Histologic examination of the tissues was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (liver, kidneys, hematopoietic/lymphoid system, nasal tissue-pharynx, spleen, thymus, thyroid gland, cecum, colon, duodenum, ileum, jejunum, and mesenteric lymph node) and relevant gross lesions.
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis.

Postmortem examinations (offspring):

Thyroid Hormone Measurements:
Serum for Thyroid Hormone Analyses:
The sequence of the sample collection for all of the animals was counterbalanced across groups (e.g., control, high, middle, low) to the extent possible on each day of sample collection to control for potential confounding influences of collection timing. Blood was placed in serum separator tubes and placed on ice. Serum was separated from cells and stored in the freezer until analysis.
Serum T3 and T4 concentrations were assessed for all samples from adult males, LD 14 dams, PND 4 culled pups, and PND 13 pups. TSH was not assessed for any of the animals.

Off-Spring Necropsy:
All PND 4 culled pups and pups surviving to PND 13 were anesthetized with a mixture of isoflurane vapors and medical oxygen. While under anesthesia, blood was collected via cardiac puncture from all culled pups and at least one PND 13 male and female per litter (if possible). While under anesthesia, the animals were euthanized by decapitation. Thyroids were removed from each pup that had blood collected, and fixed for possible histopathological examination.

Statistics:

Descriptive statistics (means and standard deviations), Bartlett's test, parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA), Dunnett's test, Wilcoxon Rank-Sum test with Bonferroni's correction, Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969), Fisher exact probability test with Bonferroni's correction, the binomial distribution test, sex ratio calculations, censored Wilcoxon test, z-test of proportions. See "Other information" for complete description of statistical analysis performed.

Reproductive indices:

Reproductive indices will be calculated for all dose level groups as follows:
• Female mating index = (No. females with evidence of mating/No. paired) x 100
• Male mating index = (No. males mated/No. paired) x 100
• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
• Male conception index = (No. males siring a litter/No. mated) x 100
• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
• Male fertility index = (No. males siring a litter/No. paired) x 100
• Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100
• Gestation survival index = percentage of delivered pups alive at birth
• Post-implantation loss = (No. implants – No. offspring)/(No. implants) x 100

Offspring viability indices:

• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100
• Day 7 or 13 pup survival index = (No. viable pups on day 7 or 13/No. after culling) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

In-Life Observations:
No treatment-related effects on behavior or demeanor were observed at any dose level during the study period. There were no notable observations made during the cage-side observations (data in study file).

Detailed Clinical Observations:
Examinations performed on all rats revealed no treatment-related or statistically significant findings.

Mortality:

no mortality observed

Description (incidence):

All animals survived until termination.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were statistically identified and 7% lower on GD 20 in females given 200 mg/kg/day when compared to controls. Body weight gains were statistically identified and 14.5% lower from GD 0-20 in females given 200 mg/kg/day when compared to controls. These decreases were associated with decreased feed consumption throughout gestation at this dose level. There was a treatment-related decrease in lactation body weights (4-6% throughout lactation) in females given 200 mg/kg/day. There were no corresponding decreases in feed consumption or body weight gains during lactation in this groups, and therefore, the lower body weights likely reflects the lower body weights and body weight gains during gestation. There were no treatment-related differences in the body weights of females at any dose level tested during the pre-mating period or in body weight gains during the lactation period. There were no treatment-related differences in body weights of females in the 10 or 50 mg/kg/day groups during lactation. No significant differences in body weights were observed for males at any dose level when compared to controls.

Final Body weights:
Females given 200 mg/kg/day had a treatment-related statistically identified 8.1% lower mean final body weight, relative to controls. The final body weights of females given 10 or 50 mg/kg/day and of males given 10, 50 or 200 mg/kg/day were similar to controls (See Attachment "OECD 422 Results Tables").

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males given 200 mg/kg/day, there was a treatment-related decrease in feed consumption of 26% from TD 1-3 and 11% from TD 3-8 compared to controls. There was a treatment-related decrease in feed consumption of 43% from TD 1-3, 16% from TD 3-8, and 11% from TD 15-22 in the 200 mg/kg/day group females compared to controls. Feed consumption was decreased throughout gestation (7-9% compared to controls) in females given 200 mg/kg/day. There were no significant differences in the amount of feed consumed by males or females at 10 or 50 mg/kg/day when compared to their respective controls throughout the study (See Attachment "OECD 422 Results Tables").

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
Males and females given 200 mg/kg/day had statistically significant treatment-related higher mean reticulocyte counts. The higher reticulocyte counts were interpreted to be a regenerative response to hemolysis of the red blood cells at this dose level. The presence of hemolysis was indicated by elevations in total serum bilirubin, urine bilirubin and urine urobilinogen in males and females given 200 mg/kg/day (see Clinical Chemistry and Urinalysis sections). The hemolysis was interpreted to be primarily extravascular, as indicated by the presence of increased numbers of hemosiderin-laden macrophages in the spleens of males and females given 200 mg/kg/day, the absence of noticeable hemoglobinemia, and no significant increases in MCHC and MCH at any dose level. There were no corresponding decrements in the mean red blood cell counts or mean hematocrits of males or females given 200 mg/kg/day, which was indicative of compensated hemolytic anemia. Due to the compensated status of the hematologic alterations, the hemolysis and associated reticulocytosis were interpreted to be non-adverse effects.
Treatment-related very slight polychromasia of red blood cells, which was consistent with the increase in reticulocyte counts, was present in 5/12 males and 4/12 females given 200 mg/kg/day. The polychromasia was interpreted to be reflective of a non-adverse regenerative response to the hemolytic anemia.
Males given 200 mg/kg/day had a statistically significant treatment-related higher mean white blood cell count, and treatment-related higher percent neutrophils. There were no definitive histopathologic correlates for the higher white blood cell counts. Males given 200 mg/kg/day also had lower percent lymphocytes, which was interpreted to be reflective of the increase in percent neutrophils at this dose level. The alterations in white blood cell parameters were non-adverse (See Attachment "OECD 422 Results Tables").

Coagulation:
There were no treatment-related changes in the prothrombin times of males or females at any dose level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical Chemistry:
Males and females given 200 mg/kg/day had statistically significant treatment-related higher mean total bilirubin concentrations, which were interpreted to be caused by hemolysis (as described in the Hematology section above). Males given 200 mg/kg/day had a statistically significant treatment-related higher mean phosphorus concentration. The higher serum phosphorus may have been related to the increase in hyaline droplet formation (consistent with alpha 2u globulin) that was present in the kidneys of males given 200 mg/kg/day (see Histopathology section). Females given 200 mg/kg/day had statistically significant treatment-related higher mean cholesterol and triglyceride concentrations, which were likely the result of altered lipid metabolism due to treatment-related liver effects (as evidenced by increased liver weights and hepatocellular hypertrophy in females at this dose level – see Organ Weights and Histopathology sections). Females given 200 mg/kg/day had statistically significant lower mean albumin and glucose concentrations, a statistically significant lower alanine aminotransferase (ALT) activity, and a statistically significant higher mean potassium concentration. The alterations in albumin, glucose, ALT and potassium were interpreted to be treatment-related, based on trends that were present when comparing the values of individual animals from the 200 mg/kg/day group with individual animal values from the control group. Males given 200 mg/kg/day had statistically significant lower urea nitrogen and total protein concentrations, and females given 200 mg/kg/day had a statistically significant higher chloride concentration. The alterations in urea nitrogen, total protein and chloride concentrations were interpreted to be unrelated to treatment because the values were of minimal difference from controls, and the data lacked clear dose responsive progressions. None of the alterations in clinical chemistry parameters were interpreted to be indicative of adverse effects (See Attachment "OECD 422 Results Tables").

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males given 10, 50 or 200 mg/kg/day had the presence of bilirubin in the urine, which was interpreted to be treatment-related. The amount of bilirubin in the urine increased in a dose-responsive manner, with 10/12 low dose males having a small amount of bilirubin, 2/12 mid-dose males having a moderate amount of bilirubin, and 10/12 mid-dose and 12/12 high-dose males having a large amount of bilirubin in the urine. The increase in urine bilirubin was accompanied by a dose-responsive discoloration of the urine, with 8/12 low-dose males and 5/12 mid-dose males having dark yellow urine, 7/12 mid-dose males having brown urine, and 12/12 high-dose males having dark brown urine. The discoloration of the urine was interpreted to be caused by the presence of bilirubin. Additional treatment-related alterations in males given 200 mg/kg/day consisted of the presence of moderate amounts of protein in 10/12 animals, the presence of trace amounts of glucose in 10/12 animals, and increases in the concentration of urobilinogen (1.0 EU/dL in 7/10 animals and 2.0 EU/dL in 3/12 animals). None of the alterations in urinalysis parameters were interpreted to be indicative of adverse effects (See Attachment "OECD 422 Results Tables").

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Tests:
Sensory Evaluation:
Examinations performed on males and females revealed no treatment-related findings in the sensory evaluation. There were two observations in males that were statistically-significant when compared to control (response to tail pinch pronounced, baseline, 50 mg/kg/day, p = 0.0285; and response to tail pinch pronounced, treated, 10 mg/kg/day, p = 0.0120). These observations were not deemed related to treatment as they either occurred under baseline conditions (prior to dosing) or were not present in a dose responsive manner.

Rectal Temperature:
There were no treatment-related effects on rectal temperature either in males (p = 0.8668) or females (p = 0.1735).

Grip Performance:
There were no treatment-related effects on hindlimb grip performance either in males (p = 0.6312) or females (p = 0.0965). Similarly, there were no treatment-related effects on forelimb grip performance either in males (p = 0.8618) or females (p = 0.1999).

Motor Activity:
There were no treatment-related effects on motor activity. Treatment did not affect motor activity total counts (treatment x time interaction) either in males (p = 0.2591) or in females (p = 0.9572). The distribution of the motor activity counts within session (treatment x time x interval interaction) was not affected by treatment either in males (p = 0.3379) or in females (p = 0.3207).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related liver effect consisted of hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in males given 50 mg/kg/day and in males and females given 200 mg/kg/day. The hepatocellular hypertrophy was graded as very slight in males given 50 mg/kg/day and in females given 200 mg/kg/day, and was graded as slight in males given 200 mg/kg/day. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect, based on the modest (<25%) corresponding increases in liver weights, and the absence of any additional treatment-related liver alterations such as necrosis, increased apoptosis, inflammation, proliferative or degenerative changes in the liver of males and females at any dose level.
A treatment-related kidney effect consisted of a very slight increase in hyaline droplet formation (consistent with alpha 2u globulin) in the proximal convoluted tubules of males given 50 or 200 mg/kg/day, compared to controls. The occurrence of increased amounts of hyaline droplets was interpreted to be a non-adverse effect because of the absence of any additional treatment-related kidney alterations in males at any dose level. Hyaline droplet formation of the kidneys is a male rat specific lesion with no significant relevance to humans. The hyaline droplets were negative for iron with Prussian blue staining, and negative for bilirubin with Fouchet’s bile stain. Another treatment-related kidney effect consisted of a very slight increase in hematogenous pigment in renal tubules of females given 50 or 200 mg/kg/day. The pigment was present in the cytoplasm of cortical tubules, and was weakly positive for bilirubin with Fouchet’s bile stain, and negative for iron with Prussian blue stain. The presence of bilirubin in the renal cortical tubules was consistent with effects associated with hemolytic anemia, and was interpreted to be a non-adverse effect.
Treatment-related effects of the spleen consisted of an increase in the incidence of slight congestion of the red pulp in males and females given 200 mg/kg/day, very slight or slight increased erythrocytic extramedullary hematopoiesis in males given 50 mg/kg/day and in males or females given 200 mg/kg/day, very slight increased granulocytic extramedullary hematopoiesis in males given 200 mg/kg/day, and very slight or slight increases in pigment-laden macrophages in males and females given 10, 50 or 200 mg/kg/day. The macrophages stained positive for iron with Prussian blue stain, and were therefore consistent with the presence of hemosiderin. The congestion, erythrocytic extramedullary hematopoiesis and pigment-laden macrophages were consistent with effects associated with extravascular hemolytic anemia. There were no consistent inflammatory lesions in other tissues that corresponded to the increased myelocytic extramedullary hematopoiesis of the spleen. All of the histopathologic alterations of the spleen were interpreted to be non-adverse.
Treatment-related very slight polychromasia of red blood cells (recorded under the Hematopoietic/Lymphoid System) was present in 5/12 males and 4/12 females given 200 mg/kg/day. The polychromasia was interpreted to be reflective of a non-adverse regenerative response to the hemolytic anemia.
Treatment-related subacute to chronic inflammation in the mucosa and submucosa of the cecum was present in 4/12 males given 200 mg/kg/day. The inflammation was very slight in 2/12 males, slight in 1/12 rats, and moderate in 1/12 rats given 200 mg/kg/day. The two males that had slight or moderate inflammation of the cecum also had slight focally extensive or diffuse edema in the submucosa of the cecum. The male with moderate inflammation of the cecum also had slight, multifocal necrosis of mucosal epithelial cells. The inflammation, edema, and mucosal necrosis of the cecum were interpreted to be adverse locally irritating effects of treatment.
Treatment-related atrophy of the lymphoid tissue of the thymus was present in 8/12 females given 200 mg/kg/day. The thymic atrophy was interpreted to be a stress-induced adverse effect.
A treatment-related increase in the incidence of very slight hypertrophy of follicular cells of the thyroid gland was present in females given 200 mg/kg/day. The follicular cell hypertrophy corresponded to treatment-related decrements in T4 concentrations in females given 200 mg/kg/day. The hypertrophy was interpreted to be a non-adverse, adaptive effect, which may have been caused by induction of liver microsomal enzymes responsible for the biliary excretion of thyroid hormones, with resultant chronic stimulation of thyroid hormone production through disruption of the hypothalamic – pituitary – thyroid axis.
Treatment-related effects of the nasal tissues and/or pharyngeal duct were present in 2/12 males given 200 mg/kg/day, 1/12 females given 10 mg/kg/day, 3/12 females given 50 mg/kg/day, and 6/12 females given 200 mg/kg/day. The effects were randomly distributed within the nasal passages, and consisted of variable incidences and severities of the following: degeneration in the olfactory, respiratory, transitional and/or pharyngeal duct epithelium; fibrinopurulent exudate; chronic active inflammation in areas of epithelial degeneration; squamous metaplasia of the respiratory, transitional and/or pharyngeal duct epithelium; ulcers of the respiratory and/or pharyngeal duct epithelium, and synechia of the turbinates. All of the nasal tissue/pharyngeal effects were interpreted to be adverse and reflective of direct irritation due to inadvertent reflux of the test material into the pharyngeal duct and nasal passages during oral gavage administration.
Pigment-laden macrophages (brown pigment) were present in the mucosa and/or submucosa of the duodenum, jejunum, ileum, cecum and/or colon of several males and females given 50 or 200 mg/kg/day. Pigment-laden macrophages (brown pigment) were also present in the mesenteric lymph nodes in 1/12 females given 10 mg/kg/day, 11/12 males and 5/12 females given 50 mg/kg/day, and 12/12 males and 11/12 females given 200 mg/kg/day. The test material is brown in color. Therefore, the pigment-laden macrophages were interpreted to be reflective of absorption of the brown test material, and not an adverse effect of treatment. The pigment in the macrophages was negative for iron with Prussian blue stain, and negative for bile with Fouchet’s bile stain (See Attachment "OECD 422 Results Tables").

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone Measurements:
There were no treatment-related changes in the serum concentrations of T3 or T4 in PND 4 culled pups, PND 13 males or females, nor in adult males, at any dose level. There was a treatment-related and statistically identified decrease in the mean T4 concentration of LD 14 dams given 200 mg/kg/day. There was no treatment-related effect on T3 at any dose level or T4 concentrations at 10 or 50 mg/kg/day in LD 14 dams (See Attachment "OECD 422 Results Tables").

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no evidence of an effect on estrous cyclicity at any dose level.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no treatment-related effects at any dose level on reproductive indices, time to mating, gestation length, postimplantation loss, pup survival, or pup sex ratio. There were no treatment-related effects on litter size.

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: There were no adverse systemic effects in males at any dose level.

Remarks on result:

other: NOAEL is for systemic toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other: NOAEL is for systemic toxicity.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment-related effects at any dose level on pup survival.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male and female pup body weights were decreased on PND 4, 7 and 13 (statistically identified in females on PND 4 (AC), 7, and 13 and in males on PND 13) in litters from dams given 200 mg/kg/day. Decreases in pup body weights were associated with decreases in maternal gestation and lactation body weights and gestation body weight gains in the 200 mg/kg/day dose group. There were no treatment-related effects on body weights in pups from dams given 10 or 50 mg/kg/day (See Attachment "OECD 422 Results Tables").

Sexual maturation:

no effects observed

Description (incidence and severity):

Anogenital Distance:
There were no treatment-related effects on anogenital distance in PND 1 male or female pups at any dose level tested.

Nipple/Areolae Retention:
There were no significant, treatment-related differences in nipple/areolae retention in either male or female offspring.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Measurements:
There were no treatment-related changes in the serum concentrations of T3 or T4 in PND 4 culled pups and PND 13 males or females at any dose level.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Analytical Chemistry:

Analyses of all dosing suspensions from the initial mix revealed mean concentrations ranging from 96.1 to 97.5% of targeted concentrations. Analyses of aliquots for the low- and high-dose suspensions indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 0.5%.

Conclusions:

A no-observed-effect level (NOEL) for general toxicity could not be determined for male or female rats due to the occurrence of treatment-related effects at all dose levels. Based on adverse locally irritating effects in the cecum and nasal tissues of males given 200 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for males was 50 mg/kg/day. However, there were no adverse systemic effects in males at any dose level. Based on adverse locally irritating effects in the nasal tissues and/or pharynx of some females at all dose levels, a NOAEL for females was not determined. However, adverse systemic effects in females were stress-induced lymphoid atrophy of the thymus and decreased body weight gain, body weight and feed consumption during gestation in rats given 200 mg/kg/day. Therefore the NOAEL for systemic toxicity was 200 mg/kg/day for males, and 50 mg/kg/day for females. Based on treatment-related decreases in pup body weights at 200 mg/kg/day, the NOEL for reproductive and developmental toxicity was 50 mg/kg/day.

Executive summary:

The purpose of this study was to evaluate the potential effects on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival in rats following repeated oral gavage administration of 4-(3-(1-naphthylamino)propyl)morpholine. This study evaluated 4-(3-(1-naphthylamino)propyl)morpholine in the OECD 422 design.

Groups of 12 male and 12 female Crl:CD(SD) rats were administered 4-(3-(1-naphthylamino)propyl)morpholine in propylene glycol daily, by gavage at dose levels of 0 (control), 10, 50, or 200 mg/kg/day. Females were dosed once daily for four weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 13. Females were necropsied on postpartum day 14. Males were dosed four weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test days 59-60). Effects on general systemic toxicity, neurobehavioral activity, clinical chemistry, hematology, coagulation, thyroid hormone levels, urine parameters, gonadal function, estrous cyclicity, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, anogenital distance, nipple retention, and the presence of gross external morphological alterations were assessed in the offspring.

Male and female rats administered 10, 50, or 200 mg/kg/day 4-(3-(1-naphthylamino)propyl)morpholinehad no treatment-related effects in clinical signs or neurobehavioral endpoints when compared to control animals. There were no treatment-related effects of 4-(3-(1-naphthylamino)propyl)morpholine on reproductive function, or survival of the offspring at any dose level tested.

Gestation body weight gains were statistically identified and 14.5% lower from GD 0-20 in females given 200 mg/kg/day when compared to controls. Body weights were statistically identified and 7% lower on GD 20 in females given 200 mg/kg/day when compared to controls. These decreases were associated with decreased feed consumption throughout gestation. There was a treatment-related decrease in lactation body weights (4-6% throughout lactation) in females given 200 mg/kg/day. There were no corresponding decreases in feed consumption or body weight gains during lactation in this group, and therefore, the lower body weights likely reflect the lower body weights and body weight gains during gestation. There were no treatment-related differences in the body weights of females at any dose level tested during the pre-mating period or in body weight gains during the lactation period. There were no treatment-related differences in body weights of females in the 10 or 50 mg/kg/day groups during lactation. No significant differences in body weights were observed for males at any dose level when compared to controls.

There was a treatment-related decrease in feed consumption of 43% from TD 1-3, 16% from TD 3-8, and 11% from TD 15-22 in females given 200 mg/kg/day when compared to controls. There was treatment-related decreased feed consumption throughout gestation of 7-9% in females given 200 mg/kg/day compared to controls. In males given 200 mg/kg/day, there was a treatment-related decrease in feed consumption of 26% from TD 1-3 and 11% from TD 3-8 compared to controls. There were no treatment-related effects on feed consumption throughout lactation. There were no significant differences in the amount of feed consumed by males or females at 10 or 50 mg/kg/day when compared to their respective controls throughout the study.

There were no treatment-related effects on litter size. Male and female pup body weights were decreased on PND 4, 7 and 13 (statistically identified in females on PND 4(AC), 7, and 13 and in males on PND 13) in litters from dams given 200 mg/kg/day. Decreases in pup body weights were associated with decreases in maternal gestation body weights and body weight gains in the 200 mg/kg/day dose group. There were no treatment-related effects on pup body weights in pups from dams given 10 or 50 mg/kg/day.

Males and females given 200 mg/kg/day had treatment-related higher mean reticulocyte counts, which were interpreted to be a regenerative response to hemolysis of the red blood cells at this dose level. The hemolysis and associated reticulocytosis were interpreted to be non-adverse effects. Treatment-related very slight polychromasia of red blood cells was present in males and females given 200 mg/kg/day, and was interpreted to be reflective of a non-adverse regenerative response to the hemolytic anemia. Males given 200 mg/kg/day had a statistically significant treatment-related higher mean white blood cell count, and treatment-related higher percent neutrophils, and lower percent lymphocytes. These alterations in white blood cell parameters were non-adverse. There were no treatment-related effects on prothrombin time in male or female rats administered 10, 50, or 200 mg/kg/day.

Males and females given 200 mg/kg/day had treatment-related higher mean total bilirubin concentrations, which were interpreted to be caused by hemolysis. Males given
200 mg/kg/day had a statistically significant treatment-related higher mean phosphorus concentration. Females given 200 mg/kg/day had treatment-related higher mean cholesterol and triglyceride concentrations, lower mean albumin and glucose concentrations, lower alanine aminotransferase (ALT) activity, and a statistically significant higher mean potassium concentration. The alterations in albumin, glucose, ALT, and potassium were interpreted to be treatment related. None of the alterations in clinical chemistry parameters were interpreted to be indicative of adverse effects.

Males given 10, 50 or 200 mg/kg/day had a treatment-related and dose-responsive increase of bilirubin in the urine. Additional treatment-related alterations in males given
200 mg/kg/day consisted of the presence of moderate amounts of protein, trace amounts of glucose, and increases in the concentration of urobilinogen in the urine. None of the alterations in urinalysis parameters were interpreted to be indicative of adverse effects.

There were no treatment-related changes in the serum concentrations of T3 or T4 in PND 4 culled pups, PND 13 males or females, nor in adult males, at any dose level. There was a treatment-related and statistically identified decrease in the mean T4 concentration of LD 14 dams given 200 mg/kg/day. There was no treatment-related effect on T3 at any dose level or T4 concentrations at 10 or 50 mg/kg/day in LD 14 dams.

Females given 200 mg/kg/day had a treatment-related 8.1% lower mean final body weight, relative to controls. The final body weights of females given 10 or 50 mg/kg/day and of males given 10, 50 or 200 mg/kg/day were similar to controls.

Males given 10, 50 or 200 mg/kg/day had dose-dependent higher absolute and relative mean kidney weights, which were interpreted to be treatment related, and corresponded to the presence of very slight increased hyaline droplet formation in males given 50 or 200 mg/kg/day, and the presence of increased hematogenous pigment (consistent with bilirubin) in females given 50 or 200 mg/kg/day. Females given 200 mg/kg/day had higher mean relative kidney weights that were interpreted to be reflective of the lower mean final body weight of females at this dose level.

Males and females given 200 mg/kg/day had treatment-related higher mean absolute and relative liver weights (19.9% and 23.2% higher for males, respectively and 9.9% and 19.7% higher for females, respectively). Treatment-related higher mean absolute and relative liver weights (not statistically identified) were also present in males given 50 mg/kg/day (8.4% and 5.6% higher than controls, respectively). The higher liver weights in males and females given 200 mg/kg/day, and in males given 50 mg/kg/day, corresponded to very slight or slight hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia. Females given 50 mg/kg/day had a 5.9% increase in relative liver weight, which was interpreted to be treatment related, but lacked a histopathologic correlate.

Males given 50 or 200 mg/kg/day had treatment-related higher mean absolute and relative spleen weights. The higher spleen weights in males given 200 mg/kg/day corresponded to slight congestion of the red pulp, very slight or slight increased erythrocytic and granulocytic extramedullary hematopoiesis, and very slight or slight increased amount of pigment (hemosiderin)-laden macrophages. The higher spleen weights in males given 50 mg/kg/day corresponded to very slight increased erythrocytic extramedullary hematopoiesis, and very slight increased amount of pigment (hemosiderin)-laden macrophages.

Males given 200 mg/kg/day had treatment-related higher mean absolute and relative thyroid weights. The higher thyroid weights in males given 200 mg/kg/day did not have a histopathologic correlate, and were not accompanied by alterations in serum T3 and T4 concentrations.

Females given 200 mg/kg/day had treatment-related statistically identified lower mean absolute and relative thymus weights. The lower mean thymus weights corresponded to very slight atrophy of lymphoid tissue in the cortex of the thymus.

A treatment-related histopathologic liver effect consisted of hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in males given 50 mg/kg/day and in males and females given 200 mg/kg/day, which was interpreted to be a non-adverse and adaptive effect.

A treatment-related histopathologic kidney effect consisted of a very slight increase compared to controls in hyaline droplet formation in the proximal convoluted tubules of males given 50 or 200 mg/kg/day. The occurrence of increased amounts of amounts of hyaline droplets was interpreted to be a non-adverse effect. Another treatment-related histopathologic kidney effect was a very slight increase in hematogenous pigment in the renal tubules of females given 50 or 200 mg/kg/day. The pigment was present in the cytoplasm of cortical tubules, and was weakly positive for bilirubin with Fouchet’s bile stain, and negative for iron with Prussian blue stain. The presence of bilirubin in the renal cortical tubules was interpreted to be a non-adverse effect.

Treatment-related histopathologic effects of the spleen were the following: an increased incidence of slight congestion of the red pulp in males and females given 200 mg/kg/day; very slight or slight increased erythrocytic extramedullary hematopoiesis in males given 50 mg/kg/day and in males or females given 200 mg/kg/day; very slight increased granulocytic extramedullary hematopoiesis in males given 200 mg/kg/day; and very slight or slight increases in pigment-laden macrophages in males and females given 10, 50 or 200 mg/kg/day. The macrophages stained positive for iron with Prussian blue stain, and were therefore consistent with the presence of hemosiderin. All of the histopathologic alterations of the spleen were interpreted to be non-adverse.

Treatment-related very slight to moderate subacute to chronic inflammation in the mucosa and submucosa of the cecum was present in males given 200 mg/kg/day. Males with slight or moderate inflammation of the cecum also had slight focally extensive or diffuse edema in the submucosa of the cecum. The male with moderate inflammation of the cecum also had slight, multifocal necrosis of mucosal epithelial cells. The inflammation, edema, and mucosal necrosis of the cecum were interpreted to be adverse locally irritating effects of treatment.

Treatment-related atrophy of the lymphoid tissue of the thymus was present in females given 200 mg/kg/day. The thymic atrophy was interpreted to be a stress-induced adverse effect.

A treatment-related increase in the incidence of very slight hypertrophy of follicular cells of the thyroid gland was present in females given 200 mg/kg/day.

Treatment-related histopathologic effects of the nasal tissues and/or pharyngeal duct were present in males and females given 200 mg/kg/day, and females given 10 or 50 mg/kg/day. The effects were randomly distributed within the nasal passages, and consisted of variable incidences and severities of the following: degeneration in the olfactory, respiratory, transitional and/or pharyngeal duct epithelium; fibrinopurulent exudate; chronic active inflammation in areas of epithelial degeneration; squamous metaplasia of the respiratory, transitional and/or pharyngeal duct epithelium; ulcers of the respiratory and/or pharyngeal duct epithelium, and synechia of the turbinates. All of the nasal tissue/pharyngeal effects were interpreted to be adverse and reflective of direct irritation due to inadvertent reflux of the test material into the pharyngeal duct and nasal passages during oral gavage administration.

Pigment-laden macrophages (brown pigment) were present in the mucosa and/or submucosa of the duodenum, jejunum, ileum, cecum and/or colon of males and females given 50 or
200 mg/kg/day. Pigment-laden macrophages (brown pigment) were also present in the mesenteric lymph nodes in males given 50 or 200 mg/kg/day and females given 10, 50, or 200 mg/kg/day. The pigment-laden macrophages were interpreted to be reflective of absorption of the test material, which was brown in color, and not an adverse effect of treatment.

A no-observed-effect level (NOEL) for general toxicity could not be determined for male or female rats due to the occurrence of treatment-related effects at all dose levels. Based on adverse locally irritating effects in the cecum and nasal tissues of males given 200 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for males was 50 mg/kg/day. However, there were no adverse systemic effects in males at any dose level. Based on adverse locally irritating effects in the nasal tissues and/or pharynx of some females at all dose levels, a NOAEL for females was not determined. However, adverse systemic effects in females were stress-induced lymphoid atrophy of the thymus and decreased body weight gain, body weight and feed consumption during gestation in rats given 200 mg/kg/day. Therefore the NOAEL for systemic toxicity was 200 mg/kg/day for males, and 50 mg/kg/day for females. Based on treatment-related decreases in pup body weights at 200 mg/kg/day, the NOEL for reproductive and developmental toxicity was 50 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

good

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this study was to evaluate the potential effects on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival in rats following repeated oral gavage administration of 4-(3-(1-naphthylamino)propyl)morpholine. This study evaluated 4-(3-(1-naphthylamino)propyl)morpholine in the OECD 422 design.

Groups of 12 male and 12 female Crl:CD(SD) rats were administered 4-(3-(1-naphthylamino)propyl)morpholine in propylene glycol daily, by gavage at dose levels of 0 (control), 10, 50, or 200 mg/kg/day. Females were dosed once daily for four weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 13. Females were necropsied on postpartum day 14. Males were dosed four weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test days 59-60). Effects on general systemic toxicity, neurobehavioral activity, clinical chemistry, hematology, coagulation, thyroid hormone levels, urine parameters, gonadal function, estrous cyclicity, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, anogenital distance, nipple retention, and the presence of gross external morphological alterations were assessed in the offspring.

Male and female rats administered10, 50, or 200mg/kg/day 4-(3-(1-naphthylamino)propyl)morpholinehad no treatment-related effects in clinical signs orneurobehavioral endpoints whencompared to control animals. There were no treatment-related effects of 4-(3-(1-naphthylamino)propyl)morpholine on reproductive function, or survival of the offspring at any dose level tested.

Gestation body weight gains were statistically identified and 14.5% lower from GD 0-20 in females given 200 mg/kg/day when compared to controls. Body weights were statistically identified and 7% lower on GD 20 in females given 200 mg/kg/day when compared to controls. These decreases were associated with decreased feed consumption throughout gestation. There was a treatment-related decrease in lactation body weights (4-6% throughout lactation) in females given 200 mg/kg/day. There were no corresponding decreases in feed consumption or body weight gains during lactation in this group, and therefore, the lower body weights likely reflect the lower body weights and body weight gains during gestation. There were no treatment-related differences in the body weights of females at any dose level tested during the pre-mating period or in body weight gains during the lactation period. There were no treatment-related differences in body weights of females in the 10 or 50 mg/kg/day groups during lactation. No significant differences in body weights were observed for males at any dose level when compared to controls.

There was a treatment-related decrease in feed consumption of 43% from TD 1-3, 16% from TD 3-8, and 11% from TD 15-22 in females given 200 mg/kg/day when compared to controls. There was treatment-related decreased feed consumption throughout gestation of 7-9% in females given 200 mg/kg/day compared to controls. In males given 200 mg/kg/day, there was a treatment-related decrease in feed consumption of 26% from TD 1-3 and 11% from TD 3-8 compared to controls. There were no treatment-related effects on feed consumption throughout lactation. There were no significant differences in the amount of feed consumed by males or females at 10 or 50 mg/kg/day when compared to their respective controls throughout the study.

There were no treatment-related effects on litter size. Male and female pup body weights were decreased on PND 4, 7 and 13 (statistically identified in females on PND 4(AC), 7, and 13 and in males on PND 13) in litters from dams given 200 mg/kg/day. Decreases in pup body weights were associated with decreases in maternal gestation body weights and body weight gains in the 200 mg/kg/day dose group. There were no treatment-related effects on pup body weights in pups from dams given 10 or 50 mg/kg/day.

Males and females given 200 mg/kg/day had treatment-related higher mean reticulocyte counts, which were interpreted to be a regenerative response to hemolysis of the red blood cells at this dose level. The hemolysis and associated reticulocytosis were interpreted to be non-adverse effects. Treatment-related very slight polychromasia of red blood cells was present in males and females given 200 mg/kg/day, and was interpreted to be reflective of a non-adverse regenerative response to the hemolytic anemia. Males given 200 mg/kg/day had a statistically significant treatment-related higher mean white blood cell count, and treatment-related higher percent neutrophils, and lower percent lymphocytes. These alterations in white blood cell parameters were non-adverse. There were no treatment-related effects on prothrombin time in male or female rats administered10, 50, or
200mg/kg/day.

Males and females given 200 mg/kg/day had treatment-related higher mean total bilirubin concentrations, which were interpreted to be caused by hemolysis. Males given
200 mg/kg/day had a statistically significant treatment-related higher mean phosphorus concentration. Females given 200 mg/kg/day had treatment-related higher mean cholesterol and triglyceride concentrations, lower mean albumin and glucose concentrations, lower alanine aminotransferase (ALT) activity, and a statistically significant higher mean potassium concentration. The alterations in albumin, glucose, ALT, and potassium were interpreted to be treatment related. None of the alterations in clinical chemistry parameters were interpreted to be indicative of adverse effects.

Males given 10, 50 or 200 mg/kg/day had a treatment-related and dose-responsive increase of bilirubin in the urine. Additional treatment-related alterations in males given
200 mg/kg/day consisted of the presence of moderate amounts of protein, trace amounts of glucose, and increases in the concentration of urobilinogen in the urine. None of the alterations in urinalysis parameters were interpreted to be indicative of adverse effects.

There were no treatment-related changes in the serum concentrations of T3 or T4 in PND 4 culled pups, PND 13 males or females, nor in adult males, at any dose level. There was a treatment-related and statistically identified decrease in the mean T4 concentration of LD 14 dams given 200 mg/kg/day. There was no treatment-related effect on T3 at any dose level or T4 concentrations at 10 or 50 mg/kg/day in LD 14 dams.

Females given 200 mg/kg/day had a treatment-related 8.1% lower mean final body weight, relative to controls. The final body weights of females given 10 or 50 mg/kg/day and of males given 10, 50 or 200 mg/kg/day were similar to controls.

Males given 10, 50 or 200 mg/kg/day had dose-dependent higher absolute and relative mean kidney weights, which were interpreted to be treatment related, and corresponded to the presence of very slight increased hyaline droplet formation in males given 50 or
200 mg/kg/day, and the presence of increased hematogenous pigment (consistent with

 

bilirubin) in females given 50 or 200 mg/kg/day. Females given 200 mg/kg/day had higher mean relative kidney weights that were interpreted to be reflective of the lower mean final body weight of females at this dose level.

Males and females given 200 mg/kg/day had treatment-related higher mean absolute and relative liver weights (19.9% and 23.2% higher for males, respectively and 9.9% and 19.7% higher for females, respectively). Treatment-related higher mean absolute and relative liver weights (not statistically identified) were also present in males given 50 mg/kg/day (8.4% and 5.6% higher than controls, respectively). The higher liver weights in males and females given 200 mg/kg/day, and in males given 50 mg/kg/day, corresponded to very slight or slight hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia. Females given 50 mg/kg/day had a 5.9% increase in relative liver weight, which was interpreted to be treatment related, but lacked a histopathologic correlate.

Males given 50 or 200 mg/kg/day had treatment-related higher mean absolute and relative spleen weights. The higher spleen weights in males given 200 mg/kg/day corresponded to slight congestion of the red pulp, very slight or slight increased erythrocytic and granulocytic extramedullary hematopoiesis, and very slight or slight increased amount of pigment (hemosiderin)-laden macrophages. The higher spleen weights in males given
50 mg/kg/day corresponded to very slight increased erythrocytic extramedullary hematopoiesis, and very slight increased amount of pigment (hemosiderin)-laden macrophages.

Males given 200 mg/kg/day had treatment-related higher mean absolute and relative thyroid weights. The higher thyroid weights in males given 200 mg/kg/day did not have a histopathologic correlate, and were not accompanied by alterations in serum T3 and T4 concentrations.

Females given 200 mg/kg/day had treatment-related statistically identified lower mean absolute and relative thymus weights. The lower mean thymus weights corresponded to very slight atrophy of lymphoid tissue in the cortex of the thymus.

A treatment-related histopathologic liver effect consisted of hypertrophy of centrilobular/midzonal hepatocytes, with increased cytoplasmic eosinophilia, in males given 50 mg/kg/day and in males and females given 200 mg/kg/day, which was interpreted to be a non-adverse and adaptive effect.

A treatment-related histopathologic kidney effect consisted of a very slight increase compared to controls in hyaline droplet formation in the proximal convoluted tubules of males given 50 or 200 mg/kg/day. The occurrence of increased amounts of amounts of hyaline droplets was interpreted to be a non-adverse effect. Another treatment-related histopathologic kidney effect was a very slight increase in hematogenous pigment in the renal tubules of females given 50 or 200 mg/kg/day. The pigment was present in the cytoplasm of cortical tubules, and was weakly positive for bilirubin with Fouchet’s bile stain, and negative for iron with Prussian blue stain. The presence of bilirubin in the renal cortical tubules was interpreted to be a non-adverse effect.

Treatment-related histopathologic effects of the spleen were the following: an increased incidence of slight congestion of the red pulp in males and females given 200 mg/kg/day; very slight or slight increased erythrocytic extramedullary hematopoiesis in males given
50 mg/kg/day and in males or females given 200 mg/kg/day; very slight increased granulocytic extramedullary hematopoiesis in males given 200 mg/kg/day; and very slight or slight increases in pigment-laden macrophages in males and females given 10, 50 or
200 mg/kg/day. The macrophages stained positive for iron with Prussian blue stain, and were therefore consistent with the presence of hemosiderin. All of the histopathologic alterations of the spleen were interpreted to be non-adverse.

Treatment-related very slight to moderate subacute to chronic inflammation in the mucosa and submucosa of the cecum was present in males given 200 mg/kg/day. Males with slight or moderate inflammation of the cecum also had slight focally extensive or diffuse edema in the submucosa of the cecum. The male with moderate inflammation of the cecum also had slight, multifocal necrosis of mucosal epithelial cells. The inflammation, edema, and mucosal necrosis of the cecum were interpreted to be adverse locally irritating effects of treatment.

Treatment-related atrophy of the lymphoid tissue of the thymus was present in females given 200 mg/kg/day. The thymic atrophy was interpreted to be a stress-induced adverse effect.

A treatment-related increase in the incidence of very slight hypertrophy of follicular cells of the thyroid gland was present in females given 200 mg/kg/day.

Treatment-related histopathologic effects of the nasal tissues and/or pharyngeal duct were present in males and females given 200 mg/kg/day, and females given 10 or 50 mg/kg/day. The effects were randomly distributed within the nasal passages, and consisted of variable incidences and severities of the following: degeneration in the olfactory, respiratory, transitional and/or pharyngeal duct epithelium; fibrinopurulent exudate; chronic active inflammation in areas of epithelial degeneration; squamous metaplasia of the respiratory, transitional and/or pharyngeal duct epithelium; ulcers of the respiratory and/or pharyngeal duct epithelium, and synechia of the turbinates. All of the nasal tissue/pharyngeal effects were interpreted to be adverse and reflective of direct irritation due to inadvertent reflux of the test material into the pharyngeal duct and nasal passages during oral gavage administration.

Pigment-laden macrophages (brown pigment) were present in the mucosa and/or submucosa of the duodenum, jejunum, ileum, cecum and/or colon of males and females given 50 or
200 mg/kg/day. Pigment-laden macrophages (brown pigment) were also present in the mesenteric lymph nodes in males given 50 or 200 mg/kg/day and females given 10, 50, or 200 mg/kg/day. The pigment-laden macrophages were interpreted to be reflective of absorption of the test material, which was brown in color, and not an adverse effect of treatment.

A no-observed-effect level (NOEL) for general toxicity could not be determined for male or female rats due to the occurrence of treatment-related effects at all dose levels. Based on adverse locally irritating effects in the cecum and nasal tissues of males given
200 mg/kg/day, the no-observed-adverse-effect level (NOAEL) for males was
50 mg/kg/day. However, there were no adverse systemic effects in males at any dose level. Based on adverse locally irritating effects in the nasal tissues and/or pharynx of some females at all dose levels, a NOAEL for females was not determined. However, adverse systemic effects in females were stress-induced lymphoid atrophy of the thymus and decreased body weight gain, body weight and feed consumption during gestation in rats given 200 mg/kg/day. Therefore the NOAEL for systemic toxicity was 200 mg/kg/day for males, and 50 mg/kg/day for females. Based on treatment-related decreases in pup body weights at 200 mg/kg/day, the NOEL for reproductive and developmental toxicity was
50 mg/kg/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The only observed effect that could be considered as potential reproductive toxicity is a decreased pup weight at PND 4, 7 and 13 in the litters of the top dose group females. The decrease was statistically identified in males on PND 13 and in females on PNDs 4, 7 and 13.

These decreases in bodyweight were associated with a decrease in maternal gestation body weight and bodyweight gains and even post partum, the top dose Dams failed to gain weight back to the level of controls. Most of the Dams in the top dose group also displayed thymic atrophy which is a good indicator of generalised stress. Therefore it is reasonable to conclude that lactation could have been sufficiently compromised in the top dose dams to affect postnatal growth of the pups. i.e. it is considered that the reduction in pup bodyweight in the top dose is considered to be secondary to maternal toxicity (general).

It is possible, given the lipophylicity o the substance that it was passed through the breast milk into the pups and that reduced pup bodyweight was in some part due to general toxicity in the pups. However there are insufficient data to confirm if this is the case, and due to the very limited use of the substance as a fuel marker (at <1000ppm in automotive and heating fuel) it is considered that there is insufficient exposure potential to trigger the need for further follow up.

Justification for classification or non-classification

The affect of treatement with 200 mg/kg bw on pup bodyweight gain is considered likely to be subsequent to general maternal toxicity and stress. Therefore at this time it is concluded that criteria for classification are not met.