Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follows GLP and OECD 408
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/DUCrl
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Approximately 7 weeks
- Housing: After assignment, animals were housed two per cage in stainless steel cages. Cages had solid floors with corncob bedding and shredded aspen for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
- Diet: Animals were provided LabDiet ad libitum.
- Water: ad libitum
- Acclimation period: Upon arrival, the animals were housed two to three per cage in stainless steel cages.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: November 5,2014 To: February 3 and 4,2015
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DOSE SOLUTION PREPARATION
All dosing solutions were prepared by mixing the test material in propylene glycol (PG) at concentrations of 1.67, 5, or 16.7 mg/ml, and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Mannich base PTBP-MXDA was determined to be soluble in PG at a concentration of 250 mg/ml
- Concentration in vehicle: 1.67, 5, or 16.7 mg/ml
- Lot/batch no. (if required): MKBS5987V.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in propylene glycol from the control and all dose levels was determined pre-exposure, near the middle and end of the dosing period. The actual concentrations of test material in propylene glycol ranged from 95.8 to 106.9% of targeted values.
The homogeneity analyses were conducted on the low- and high-dose solutions concurrent with the concentration verifications. The results of the analyses indicated that the preparations were homogeneously mixed based on relative standard deviations of ≤5.8%.
Duration of treatment / exposure:
90 or 91 days
Frequency of treatment:
once daily seven days/week for at least 90 days
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected were based on the results of the previous 28-day study (Stebbins et al., 2013). As described in the previous toxicity section, males given 150 mg/kg/day had decrements in body weight gain (28%) and feed consumption (15%) at study termination compared to controls. Males and females given 150 mg/kg/day had grossly thickened nonglandular mucosa of the stomach with associated hyperkeratosis and hyperplasia, that was considered point of contact irritancy from direct exposure to the test material. While females given 150 mg/kg/day had only slight decrements in body weight gain (7%) compared to controls, histopathologic effects in the stomach of females were more severe than than at of males and included chronic or chronic-active inflammation of the nonglandular submucosa of the stomach. Based on body weight gain decrements in males and stomach irritation in females following 28 days of exposure, a dose level of 150 mg/kg/day would not have been well tolerated in the context of a 90-day study.
The high dose of 100 mg/kg/day was expected to induce signs of toxicity, such as stomach irritation, decreased body weight/body weight gain, and decreased feed consumption. The mid- and low-dose levels were expected to provide dose-response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a NOEL.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once a day, approximately at the same time each day (usually in the morning).


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. The DCO was conducted on all animals, at approximately the same time each day according to an established format.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed pre-exposure, twice during the first week, and weekly thereafter during the dosing period. Body weight gains were calculated relative to day 1.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all animals were examined by a veterinarian pre-exposure and prior to the scheduled necropsy using indirect ophthalmoscopy. One drop of 0.5% tropicamide ophthalmic solution was instilled in each eye to produce mydriasis prior to the indirect ophthalmic examinations. Eyes were also examined by a prosector during the necropsy using a moistened glass slide pressed to the cornea.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 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.
- Anaesthetic used for blood collection: Yes (identity) mixture of isoflurane vapors and medical oxygen
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were obtained from all animals 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and near the end of the treatment period for all animals.
- Battery of functions tested: sensory activity / grip strength / motor activity / rectal temperature:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption is addressed below) were first tested for equality of variance using Bartlett's test (alpha = 0.01; Winer, 1971). The raw data and transforms of log, inverse, and square root were examined in that order and appropriate transformations were made following a significant Bartlett’s test. The data were then subjected to the appropriate parametric analysis as described below.
In-life body weights were evaluated using a repeated measures (RM) analysis of variance (ANOVA), the multivariate approach, for time (the repeated factor), sex, and dose (Winer, 1971). In the RM-ANOVA, differences between the groups were primarily detected by the time-dose interaction. The first examination in the RM-ANOVA was of the time-sex-dose interaction. If significant at alpha = 0.02, the analysis was repeated separately for each sex without examining the results of other factors. The time-dose interaction was examined next. If the time-dose interaction was statistically significant at alpha = 0.05, linear contrasts
tested the time-dose interaction for the comparisons of each dose group to the control group. A Bonferroni correction was applied to the alpha level to compensate for the multiple comparisons with the control group (Miller, 1966). This correction controls the experiment-wise error rate. The corrected comparison-wise error rate of alpha = 0.02 was reported so direct comparison could be made to the Pillai’s Trace p-values generated.Terminal body weight, organ weight (absolute and relative, excluding ovaries, uterus, epididymides, and testes), urine volume, urine specific gravity, hematologic parameters, (excluding RBC indices and differential WBC counts), coagulation, and clinical chemistry parameters (excluding globulin and albumin/globulin ratio), were evaluated using a two-way ANOVA (Steel and Torrie, 1960) with the factors of sex and dose.
Clinical signs:
no effects observed
Description (incidence and severity):
All animals survived the 90-day test period. There were no treatment-related effects on detailed clinical observations. Examinations performed prior to treatment on TD 1 and weekly through TD 90 revealed all animals were within normal limits.
Mortality:
no mortality observed
Description (incidence):
All animals survived the 90-day test period. There were no treatment-related effects on detailed clinical observations. Examinations performed prior to treatment on TD 1 and weekly through TD 90 revealed all animals were within normal limits.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 100 mg/kg/day had treatment-related, statistically significant (males only) decreases in body weights that, at the end of the study (TD 90), were 12.5 and 7.5% lower than controls, respectively.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Differences were considered to be unrelated to treatment as the differences were minimal, there was a lack of a dose-response relationship, and there was no corresponding effect on body weight or body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Observations of persistent hyaloid artery, periocular soiling and reddened membrane were interpreted to be unrelated to treatment due to their presence at pre-exposure, their low incidence, and/or lack of a dose-response.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related hematologic effects for males or females at any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 100 mg/kg/day had treatment-related, statististically significant increases in urea nitrogen concentration. The higher urea nitrogen was likely related to the vacuolization of renal tubules that was present in the 100 mg/kg/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in the urinalysis parameters for male and female rats at any dose level.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related effects on sensory evalutation, rectal temperature, grip performance, or motor activity.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross pathologic observations.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: All animals survived the 90-day test period. There were no treatment-related effects on detailed clinical observations. Examinations performed prior to treatment on TD 1 and weekly through TD 90 revealed all animals were within normal limits.

BODY WEIGHT AND WEIGHT GAIN: Males and females given 100 mg/kg/day had treatment-related, statistically significant (males only) decreases in body weights that at the end of the study (TD 90) were 12.5 and 7.5% lower than controls, respectively. From TD 15-90, body weight gains of males and females given 100 mg/kg/day were also decreased. At study termination, the mean body weight gains of males and females given 100 mg/kg/day were 24.2 and 16.2% lower than controls, respectively. Body weights and body weight gains of males and females given 10 or 30 mg/kg/day were similar to controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Males and females given 100 mg/kg/day had treatment-related, statistically significant decreases in mean feed consumption values compared to controls throughout the course of the study (excluding males from TD 1-4). Decreases in feed consumption ranged from
5.7 to 15.6 %, and on TD 90 were 15.3 and 11.0% lower in high-dose males and females compared to controls, respectively. There were no treatment-related differences in feed consumption for males and females given 10 or 30 mg/kg/day compared to controls. There was an isolated incidence of a statistically significant, slightly higher feed consumption in males given 30 mg/kg/day from TD 1-4 compared to controls. Females given 10 mg/kg/day had several occurrences of statistically significant, slightly lower feed consumption during the treatment period as compared to controls. These differences were considered to be unrelated to treatment as the differences were minimal, there was a lack of a dose-response relationship, and there was no corresponding effect on body weight or body weight gain.

OPHTHALMOSCOPIC EXAMINATION: Pre-exposure examination on rats placed on study indicated that all rats were within normal limits at pre-exposure with the exception of three males and one female with persistent hyaloid artery. This observation represents a normal variation; therefore, these animals were considered acceptable for study purposes. Ophthalmologic observations of persistent hyaloid artery, periocular soiling and reddened membranes were noted prior to study termination. These observations were interpreted to be unrelated to treatment due to their presence at pre-exposure, their low incidence, and/or lack of a dose-response.

HAEMATOLOGY: There were no treatment-related hematologic effects for males or females at any dose level. Males and females given 100 mg/kg/day had statistically significant lower hemoglobin concentrations, and statistically significant higher platelet counts as compared to controls. The lower hemoglobin concentrations and higher platelet counts were interpreted to be unrelated to treatment because the values were within or near the historical control ranges of recently conducted 90-day rat studies.

CLINICAL CHEMISTRY: Males and females given 100 mg/kg/day had treatment-related, statististically significant increases in urea nitrogen concentration. The higher urea nitrogen was likely related to the vacuolization of renal tubules that was present in all males and females given 100 mg/kg/day. Males given 100 mg/kg/day had statistically significant lower alanine aminotransferase and alkaline phosphatase activities that were interpreted to be treatment related. The lower alanine aminotransferase and alkaline phosphatase activities may have been reflective of nutritional status, associated with lower feed consumption and lower body weight gain in males at this dose level, relative to controls. Males given 100 mg/kg/day had a treatmentrelated statistically significant higher aspartate aminotransferase activity, which may have been related to the Kupffer cell hypertrophy of the liver in males at this dose level. Females given 100 mg/kg/day had treatment-related, statistically significant increases in alanine aminotransferase and aspartate aminotransferase activities. The higher alanine aminotransferase and aspartate aminotransferase activities corresponded with the presence of treatment-related, very slight multifocal necrosis of individual hepatoctyes in all females given 100 mg/kg/day. Males and females given 100 mg/kg/day had statistically significant, minor decreases in total protein, cholesterol, and calcium concentrations, and statistically significant, minor increases in chloride concentrations. Males given 100 mg/kg/day also had statistically significant, minor decreases in albumin, and triglyceride concentrations. The alterations in cholesterol, calcium, chloride, total protein, albumin, and triglyceride concentrations were interpreted to be unrelated to treatment because the values were within or near the historical control ranges of recently conducted 90-day studies in Fischer 344 rats.

URINALYSIS: There were no treatment-related alterations in the urinalysis parameters for male and female rats at any dose level. Males and females given
100 mg/kg/day had statistically significant lower urine volumes, and males and females given 30 mg/kg/day had statistically significant higher urine specific
gravities. The lower urine volumes in males and females given 100 mg/kg/day were interpreted to be unrelated to treatment because the values were within
historical control ranges of recently conducted 90-day studies in Fischer 344 rats. The higher urine specific gravities in males and females given 30 mg/kg/day were interpreted to be unrelated to treatment due to the lack of a dose-response for this parameter in both sexes, and the female specific gravity value at 30 mg/kg/day was within the historical control range. All other urinalysis parameters were interpreted to be unaffected by treatment due to the lack of any clear doseresponsive trends.

Functional Tests:
Sensory Evaluation: There were no treatment-related effects on sensory evaluation. Examinations performed on males and females at termination revealed no treatment-related findings.
Rectal Temperature: There were no treatment-related effects on rectal temperature. The Treatment x Sex interaction was not significant (p = 0.1654), i.e., there was no statistically significant difference in rectal temperature across sexes with treatment. Furthermore, the analysis also revealed no statistically significant Treatment main effect (p = 0.3137), i.e., with male and female data considered together, treatment did not affect rectal temperature in rats.
Grip Performance: There were no treatment-related effects on grip performance. The Treatment xSex interaction was not significant for hindlimb (p = 0.7643) or forelimb (p =0.3700) grip performance, i.e., there was no statistically significant difference in grip performance across sexes with treatment. Furthermore, the analysis also revealed no statistically significant Treatment main effect for hindlimb (p = 0.4335) or forelimb (p = 0.2811) grip performance, i.e., with male and female data considered together, treatment did not affect grip performance in rats.
Motor Activity: There were no treatment-related effects on motor activity. The Treatment x Time x Sex interaction was significant (p = 0.0041), i.e., there was a statistically significant difference in motor activity across time points and/or sexes with treatment. The statistical analyses were therefore conducted separately for each sex. The triple interaction of Treatment x Time x Epoch was not statistically significant in males (p = 0.7241) or females (p = 0.1792), which indicated that the distribution of motor activity counts within each session was not affected by treatment.
However, the analysis revealed a statistically significant Treatment xTime interaction for both males (p = 0.0032) and females (p = 0.0041), i.e., with male
and female data considered separately, treatment affected motor activity across time points.
For males, the results of the subsequent linear contrasts for each of the group comparisons are as follows: Time x 0 vs. 10 mg/kg/day, p = 0.8643; Time x 0 vs. 30 mg/kg/day, p = 0.0064; and Time x 0 vs. 100 mg/kg/day, p = 0.4002. Only the comparison of the control and the mid-dose group was significant at alpha = 0.02. Inspection of the motor activity data indicated that there were no treatment-related effects on motor activity counts at any time point for any treated group in males. Based on the data and the lack of a dose-response relationship in the linear contrasts of the statisitical analysis, the significant interaction identified in males was considered spurious and was interpreted to be due to normal variability. For females, the results of the subsequent linear contrasts for each of the group comparisons are as follows: Time x 0 vs. 10 mg/kg/day, p = 0.3041; Time x 0 vs. 30 mg/kg/day, p = 0.0530; and Time x 0 vs. 100 mg/kg/day, p = 0.0005. Only the comparison of the control and the high-dose group was significant at alpha = 0.02. Inspection of the motor activity data indicated that there were no treatment-related effects on motor activity counts at any time point for any treated group in females. The reason for the significant Treatment x Time interaction was thought to be due to the fact that under baseline conditions (prior to treatment start), there was a 25% difference (increase) in motor activity in the high-dose females compared to the control females. Following treatment, there was an 8% difference (decrease) in total motor activity of high-dose females compared to controls. Based on the data, the significant interaction in females was considered spurious and was interpreted to be due to normal variability which was greater under baseline conditions than after treatment.

ORGAN WEIGHTS: Males given 100 mg/kg/day had a treatment-related, statistically significant lower mean final fasted body weight (12.6% lower than controls). Males given 100 mg/kg/day had statistically significant, higher absolute and relative mean kidney weights, which were interpreted to be treatment related and correlated with the histopathologic observation of very slight or slight vacuolization of cortical tubules in the kidneys of all males at this dose level. Males given 100 mg/kg/day had higher mean relative adrenal gland, liver, brain, testes, thyroid, and epididymides weights, and lower absolute heart, spleen, and thymus weights that were statistically significant and interpreted to be reflective of the lower body weights of males at this dose level. Males given 30 mg/kg/day had a statistically significant lower mean relative heart weight that was interpreted to be unrelated to treatment due to the lack of a dose-response for this parameter. Females given 100 mg/kg/day had a treatment-related, statistically significant lower mean final fasted body weight (7.8% lower than controls). Females given 100 mg/kg/day had statistically significant higher absolute and relative mean kidney weights, which were interpreted to be treatment related and correlated with the histopathologic observation of slight vacuolization of cortical tubules in the kidneys of all females at this dose level. Females given 100 mg/kg/day had treatment-related statistically significant lower mean absolute and relative uterus weights, which corresponded to the histopathologic observation of very slight or slight diffuse atrophy of the uterus in 9/10 females at this dose level. Females given 100 mg/kg/day had higher mean relative liver, brain, and thyroid weights, and lower absolute heart, ovaries, spleen, and thymus weights that were statistically significant, and interpreted to be reflective of the lower body weights of females at this dose level. Females given 30 mg/kg/day had a statistically significant lower mean relative heart weight that was interpreted to be unrelated to treatment, due to the lack of a dose-response for this parameter.

GROSS PATHOLOGY: There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with oral gavage administration of Mannich base PTBP-MXDA.

HISTOPATHOLOGY: NON-NEOPLASTIC: There were no histopathologic effects for males or females in the 10 mg/kg/day group. Males and females given 30 or 100 mg/kg/day had treatment-related histiocytosis of the lamina propria of the duodenum, jejunum, and ileum. The histiocytosis was characterized by the presence of numerous macrophages in the lamina propria of the villi. The macrophages had abundant foamy cytoplasm. In general, the histiocytosis was of greater severity in males and females given 100 mg/kg/day, and this alteration was most prominent in the duodenum and jejunum, with lesser severity in the ileum. Males and females given 30 or 100 mg/kg/day had treatment-related sinus histiocytosis of the mesenteric lymph nodes, which was more severe in high-dose males and females. The sinuses of the mesenteric lymph nodes contained numerous macrophages that had abundant foamy cytoplasm. The histiocytosis of the intestines and of the mesenteric lymph nodes was interpreted to be a portal of entry effect, representing the presence of absorbed test material in the cytoplasm of the macrophages. Males and females given 100 mg/kg/day had treatment-related slight diffuse histiocytosis of the spleen, which was characterized by numerous macrophages with abundant foamy cytoplasm, mostly located in the marginal zones of the white pulp of the spleen. A few macrophages with abundant foamy cytoplasm were also present in the red pulp of the spleen. Males and females given 100 mg/kg/day had treatment-related very slight or
slight hypertrophy of Kupffer cells. The hypertrophic Kupffer cells were present throughout the liver and had abundant foamy cytoplasm. Additional treatmentrelated effects in the liver consisted of very slight multifocal necrosis (with or without inflammation) of individual hepatocytes in all females given 100 mg/kg/day, and increases in the incidence of very slight or slight aggregates of macrophages – histiocytes, and very slight aggregates of mononuclear cells, in
females given 30 or 100 mg/kg/day. The necrotic individual hepatocytes were present in random locations throughout the liver, and were frequently
accompanied by small aggregates of lymphocytes and macrophages. All males and females given 100 mg/kg/day had very slight or slight multifocal vacuolization of renal tubular epithelial cells. The vacuolization was characterized by clear round vacuoles in the cytoplasm of numerous tubular epithelial cells in the cortex of the kidneys. Four females given 100 mg/kg/day had very slight, multifocal, unilateral or bilateral degeneration of the olfactory epithelium of the nasal tissue that was interpreted to be treatment-related. The degeneration of the olfactory epithelium was present in the anterior portion of the dorsal meatus and/or nasoturbinates, and was characterized by multifocal areas of thinning of the apical cytoplasm and disarray of the olfactory epithelial cells. Nine females given 100 mg/kg/day had treatment-related very slight or slight diffuse atrophy of the cervix, uterus, and vagina. The atrophy of the uterus was characterized by diffuse decreased thickness of the endometrial mucosa, endometrial stroma, and tunica muscularis, and corresponded to the lower mean absolute and relative uterine weights at this dose level. The atrophy of the cervix and vagina was characterized by diffuse decreased thickness of the mucosa and fibromuscular wall of these tissues.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Hematology Differences:

 Sex  Male            
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Hemoglobin (g/dL)  14.9 -16.7  15.5  15.3  15.2  15.0*
 Platelet Count (E3/uL  614 -905  591  602  610  716a
 Sex  Females            
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Hemoglobin (g/dL)  13.5 -16.7  14.9  14.7  14.8  14.8*
 Platelet Count (E3/uL)  694 -1019  627  638  599  688a

@Historical control data obtained from one 90-day oral gavage study and twelve 90-day dietary studies conducted in this laboratory from 2010 through 2015.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

a Statistically different from control mean, males and females analyzed separately, by Dunnett’s test, alpha = 0.05.

Clinical Chemistry Differences:

 Sex  Males            
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Urea Nitrogen (mg/dL)  12 -20  17  17  17  21*
 Alanine aminotransferase (U/L)  43 – 69  39  39  36  34a
 Alkaline Phosphatase (U/L)  75 -178  77  79  76  62a
 Aspartate Aminotransferase (U/L)  76 -128  78  80  81  221a
 Total Protein (g/dL)  6.2 -7.4  6.9  7.0  6.8  6.4*
 Albumin (g/dL)  3.9 -4.6  4.7  4.7  4.6  4.3a
 Cholesterol (mg/dL)  52 -74  73  71  69  65*
 Triglycerides (mg/dL)  40 -159  73  69  56  47a
 Calcium (mg/dL)  10.6 -12.5  11.2  11.3  11.3  11.0*
 Chloride (mmol/L)  97 -103  98  98  99  99*
 Sex  Females            
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Urea Nitrogen (mg/dL)  14 -19  19  19  19  22*
 Alanine Aminotransferase (U/L)  32 -67  32  32  34  45*
 Aspartate Aminotransferase (U/L)  79 -103  79  77  88  180a
 Total Protein (g/dL)  6.2 -7.4  6.1  6.2  6.0  5.9*
 Cholesterol (mg/dL)  65 -93  66  66  67  63*
 Calcium (mg/dL)  10.5 -12.7  10.5  10.6  10.5  10.4*
 Chloride (mmol/L)  100 -103  100  101  100  101*

@Historical control data obtained from one 90-day oral gavage study and twelve 90-day dietary studies conducted in this laboratory from 2010 through 2015.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

a Statistically different from control mean, males and females analyzed separately, by Dunnett’s test, alpha = 0.05.

Bold type indicates the effects were interpreted to be treatment-related.

Urinalysis Differences

 Sex  Males            
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Urine volume (mL)  2.3 -4.9  5.7  6.1  5.2  4.0*
 Urine Specific Gravity  1.066 -1.091  1.045  1.045  1.051*  1.049
 Sex  Females        
 Dose (mg/kg/day)  Historical Control@  0  10  30  100
 Urine Volume (mL)  2.4 -5.5  4.6  4.2  3.6  3.2*
 Urine Specific Gravity  1.046 -1.080  1.046  1.048  1.058*  1.054

 @Historical control data obtained from one 90-day oral gavage studies and twelve 90-day dietary studies conducted in this laboratory from 2010 through 2015.

* Statistically different from control mean, males and females analyzed together, by Dunnett’s test, alpha = 0.05.

Conclusions:
Males and females given 30 mg/kg/day had portal of entry altertations in the intestines and in mesenteric lymph nodes which were considered non-adverse. Females given 30 mg/kg/day also had treatment-related inflammatory changes (aggregates of macrophages – histiocytes and aggregates of mononuclear cells) in the liver, which were considered nonadverse. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for F344/DuCrl rats of either sex was interpreted to be 30 mg/kg/day.
Executive summary:

There were no treatment-related alterations in cage-side or clinical observations, weekly detailed clinical observations, ophthalmic examinations, functional tests, hematology, prothrombin time, or urinalysis parameters.

Males and females given 100 mg/kg/day had treatment-related decreases in body weights which were 12.5 and 7.5% lower than controls, respectively, at the end of the study. From TD 15-90, body weight gains of males and females given 100 mg/kg/day were also decreased and at study termination were 24.2 and 16.2% lower than controls, respectively. Males and females given 100 mg/kg/day had treatment-related decreases in mean feed consumption values compared to controls throughout the course of the study (5.7 to 15.6 %). There were no treatment-related effects on body weights, body weight gains, or feed consumption of males and females given 10 or 30 mg/kg/day.

Treatment-related clinical chemistry effects were limited to the 100 mg/kg/day dose group. Males and females given 100 mg/kg/day had treatment-related statistically significant increases in urea nitrogen concentration. The higher urea nitrogen was likely related to the

vacuolization of renal tubules that was present in all males and females given 100 mg/kg/day. Males given 100 mg/kg/day had statistically significant lower alanine aminotransferase and alkaline phosphatase activities that may have been associated with

lower feed consumption and lower body weight gain in males at this dose level. Males given 100 mg/kg/day had a treatment-related statistically significant higher aspartate aminotransferase activity, which may have been related to the Kupffer cell hypertrophy of

the liver in males at this dose level. Females given 100 mg/kg/day had treatment-related statistically significant increases in alanine aminotransferase and aspartate aminotransferase activities. The higher alanine aminotransferase and aspartate aminotransferase activities corresponded with the presence of treatment-related very slight multifocal necrosis of individual hepatocytes in all females given 100 mg/kg/day.

Treatment-related organ weight effects were limited to the 100 mg/kg/day dose group. Males and females given 100 mg/kg/day had statistically significant higher absolute and relative mean kidney weights which were interpreted to be treatment related, and correlated

with the histopathologic observation of very slight or slight vacuolization of cortical tubules in the kidneys of all males and females at this dose level. Females given 100 mg/kg/day had treatment-related statistically significant lower mean absolute and relative uterus weights, which corresponded to the histopathologic observation of very slight or slight diffuse atrophy of the uterus in 9/10 females at this dose level. Finally, males and females given 100 mg/kg/day had numerous statistically significant organ weight alterations that were

deemed secondary to lower terminal body weights.

Treatment-related histopathologic effects in males and females given 100 mg/kg/day consisted of histiocytosis (accumulation of macrophages that had abundant foamy cytoplasm) in the duodenum, jejunum, and ileum, and in mesenteric lymph nodes and

spleen, hypertrophy of Kupffer cells in the liver, and vacuolization of tubular epithelial cells in the cortex of the kidneys. Additional treatment-related effects in females given 100 mg/kg/day consisted of very slight multifocal necrosis (with or without inflammation)

of individual hepatocytes in random locations throughout the liver, increases in the incidence of very slight or slight aggregates of macrophages – histiocytes, and very slight aggregates of mononuclear cells in the liver, very slight multifocal degeneration of the

olfactory epithelium of the nasal tissues, and very slight or slight diffuse atrophy of the cervix, uterus, and vagina. Treatment-related histopathologic effects in males and females given 30 mg/kg/day consisted of histiocytosis of the duodenum, jejunum, ileum, and of the

mesenteric lymph nodes. Additional treatment-related effects in females given 30 mg/kg/day consisted of increases in the incidence of very slight or slight aggregates of macrophages – histiocytes and very slight aggregates of mononuclear cells in the liver. The

histiocytosis of the intestines and of mesenteric lymph nodes observed at 30 and 100 mg/kg/day was interpreted to be a portal of entry effect, representing the presence of absorbed test material in the cytoplasm of the macrophages. There were no treatmentrelated

effects in males or females given 10 mg/kg/day.

Males and females given 30 mg/kg/day had portal of entry altertations in the intestines and in mesenteric lymph nodes which were considered non-adverse. Females given 30 mg/kg/day also had treatment-related inflammatory changes (aggregates of macrophages

– histiocytes and aggregates of mononuclear cells) in the liver, which were considered non-adverse. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for F344/DuCrl rats of either sex was interpreted to be 30 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
90 day GLP/Guideline study was conducted. Groups of 10 male and female F344/DuCrl rats were administered Mannich Base PTBP-MXDA by gavage at target doses of 0,10,30, and 100 mg/kg/day. Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for F344/DuCrl rats of either sex was interpreted to be 30 mg/kg/day.

28 day GLP/Guideline study was conducted. Groups of five male and five female F344/DuCrl rats were administered Mannich Base PTBP-MXDA by gavage at targeted dose levels of 0, 15, 50, or 150 mg/kg/day in PG for 28 days to evaluate the potential for systemic toxicity. The only treatment-related effects in males and females given 50 mg/kg/day were interpreted to be portal of entry altertations in the intestines and mesenteric lymph nodes, and localized irritancy of the stomach. Therefore, the NOEL for systemic toxicity was determined to be 50 mg/kg/day in both sexes.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A subchronic repeated dose toxicity study was conducted according to OECD Guideline 408 and according to GLP.

Groups of 10 male and female F344/DuCrl rats were administered Mannich Base PTBP-MXDA by gavage at target doses of 0,10,30, and 100 mg/kg/day.

There were no treatment-related alterations in cage-side or clinical observations, weekly detailed clinical observations, ophthalmic examinations, functional tests, hematology, prothrombin time, or urinalysis parameters.

Males and females given 100 mg/kg/day had treatment-related decreases in body weights which were 12.5 and 7.5% lower than controls, respectively, at the end of the study. From TD 15-90, body weight gains of males and females given 100 mg/kg/day were also decreased and at study termination were 24.2 and 16.2% lower than controls, respectively. Males and females given 100 mg/kg/day had treatment-related decreases in mean feed consumption values compared to controls throughout the course of the study (5.7 to 15.6 %). There were no treatment-related effects on body weights, body weight gains, or feed consumption of males and females given 10 or 30 mg/kg/day.

Treatment-related clinical chemistry effects were limited to the 100 mg/kg/day dose group. Males and females given 100 mg/kg/day had treatment-related statistically significant increases in urea nitrogen concentration. The higher urea nitrogen was likely related to the vacuolization of renal tubules that was present in all males and females given 100 mg/kg/day. Males given 100 mg/kg/day had statistically significant lower alanine aminotransferase and alkaline phosphatase activities that may have been associated with lower feed consumption and lower body weight gain in males at this dose level. Males given 100 mg/kg/day had a treatment-related statistically significant higher aspartate aminotransferase activity, which may have been related to the Kupffer cell hypertrophy of the liver in males at this dose level. Females given 100 mg/kg/day had treatment-related statistically significant increases in alanine aminotransferase and aspartate aminotransferase activities. The higher alanine aminotransferase and aspartate aminotransferase activities corresponded with the presence of treatment-related very slight multifocal necrosis of individual hepatocytes in all females given 100 mg/kg/day.

Treatment-related organ weight effects were limited to the 100 mg/kg/day dose group. Males and females given 100 mg/kg/day had statistically significant higher absolute and relative mean kidney weights which were interpreted to be treatment related, and correlated with the histopathologic observation of very slight or slight vacuolization of cortical tubules in the kidneys of all males and females at this dose level. Females given 100 mg/kg/day had treatment-related statistically significant lower mean absolute and relative uterus weights, which corresponded to the histopathologic observation of very slight or slight diffuse atrophy of the uterus in 9/10 females at this dose level. Finally, males and females given 100 mg/kg/day had numerous statistically significant organ weight alterations that were deemed secondary to lower terminal body weights.

Treatment-related histopathologic effects in males and females given 100 mg/kg/day consisted of histiocytosis (accumulation of macrophages that had abundant foamy cytoplasm) in the duodenum, jejunum, and ileum, and in mesenteric lymph nodes and spleen, hypertrophy of Kupffer cells in the liver, and vacuolization of tubular epithelial cells in the cortex of the kidneys. Additional treatment-related effects in females given 100 mg/kg/day consisted of very slight multifocal necrosis (with or without inflammation) of individual hepatocytes in random locations throughout the liver, increases in the incidence of very slight or slight aggregates of macrophages – histiocytes, and very slight aggregates of mononuclear cells in the liver, very slight multifocal degeneration of the olfactory epithelium of the nasal tissues, and very slight or slight diffuse atrophy of the cervix, uterus, and vagina. Treatment-related histopathologic effects in males and females given 30 mg/kg/day consisted of histiocytosis of the duodenum, jejunum, ileum, and of the mesenteric lymph nodes. Additional treatment-related effects in females given 30 mg/kg/day consisted of increases in the incidence of very slight or slight aggregates of macrophages – histiocytes and very slight aggregates of mononuclear cells in the liver. The histiocytosis of the intestines and of mesenteric lymph nodes observed at 30 and 100 mg/kg/day was interpreted to be a portal of entry effect, representing the presence of absorbed test material in the cytoplasm of the macrophages. There were no treatment-related effects in males or females given 10 mg/kg/day.

Males and females given 30 mg/kg/day had portal of entry alterations in the intestines and in mesenteric lymph nodes which were considered non-adverse. Females given 30 mg/kg/day also had treatment-related inflammatory changes (aggregates of macrophages – histiocytes and aggregates of mononuclear cells) in the liver, which were considered non-adverse. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for F344/DuCrl rats of either sex was interpreted to be 30 mg/kg/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Males and females given 30 mg/kg/day had portal of entry alterations in the intestines and in mesenteric lymph nodes which were considered non-adverse. Females given 30 mg/kg/day also had treatment-related inflammatory changes (aggregates of macrophages – histiocytes and aggregates of mononuclear cells) in the liver, which were considered non-adverse. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for F344/DuCrl rats of either sex was interpreted to be 30 mg/kg/day.

Justification for classification or non-classification

Evidence was conclusive but not sufficient for classification.