Registration Dossier

Administrative data

Description of key information

Results of a 28 day oral gavage study are available. Effects were noted in the stomach (considered point of contact irritant effects) and kidney.

Results of a 90 day oral gavage study are available. No adverse effects were observed, although male body weight gain was significantly decreased on test day 8.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline Study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
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 6 weeks
- Weight at study initiation: Males: 114.3-138.2 g; Females: 86.1-100.5 g
- Fasting period before study: no
- Housing: one per cage in stainless steel cages. Cages had solid floors with corncob bedding and shredded Aspen bedding
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form ad libitum
- Water (e.g. ad libitum): municipal water was provided ad libitum
- Acclimation period: approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour
- 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: October 11, 2012 To: November 8, 2012
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
All dosing solutions were prepared by mixing the test material in propylene glycol (PG) at concentrations of 0, 16.7, 50, or 166.7 mg/ml and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted at least weekly based on individual body weights.

VEHICLE
- Concentration in vehicle: 0, 16.7, 50, or 166.7 mg/ml
- Amount of vehicle (if gavage): 6 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation analyses of all dose solutions were determined pre-exposure. The homogeneity of the low-dose and the high-dose solutions were determined concurrent with dose confirmation. The method used for analyzing the test material in propylene glycol was high performance liquid chromatography with ultraviolet detection (HPLC/UV).
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300, or 1000 MKD
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels selected are shown in Text Table 1. The high dose was the limit dose of 1000 mg/kg/day. 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 no-observed-effect level (NOEL).
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table 1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure and once per week throughout the study

BODY WEIGHT: Yes
- Time schedule for examinations: pre-exposure, twice during the first week and at least weekly during the dosing period

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and pre-terminal
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: terminal
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: terminal
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the week prior to the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [4] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5)
HISTOPATHOLOGY: Yes (see table 6)
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. 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. 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. Terminal body weight, organ weight (absolute and relative, excluding epididymides, testes, prostate+seminal vesicles with coagulating glands (and fluids), and prostate weights), 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. Results for epididymides, testes, prostate + seminal vesicles with coagulating glands (and fluids), and prostate weights (absolute and relative) were analyzed using a one-way ANOVA. Feed consumption data were evaluated by Bartlett's test for equality of variances. Descriptive statistics only (means and standard deviations) were reported for body weight gains, globulin, albumin/globulin ratio, RBC indices, and differential WBC counts. Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (type I errors) was greater than the nominal alpha levels.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 2
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 3
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 4
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Table 7
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 5
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 6
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All rats survived the full duration of the study.
Examinations performed on all animals revealed no treatment-related findings.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically identified or treatment-related differences in the body weights or body weight gains of male or female rats at any dose level as compared to their respective controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no statistically identified or treatment-related differences in the feed consumption of male or female rats at any dose level as compared to their respective controls.

OPHTHALMOSCOPIC EXAMINATION
Pre-exposure examination on all animals placed on study indicated all but two rats were within normal limits. These two males assigned to the 300 mg/kg/day group had cloudy corneas during the pre-exposure examination, but were considered suitable for study purposes.
In addition to pre-exposure observations, cloudy cornea was noted in four male rats one in the 100 mg/kg/day group, two in the 300 mg/kg/day group, and one in the 1000 mg/kg/day group) and three female rats (two in the 100 mg/kg/day group, and one in the 300 mg/kg/day group) prior to study termination. These observations were interpreted to be unrelated to treatment due to their low incidence and lack of a dose-response relationship.

HAEMATOLOGY
Treatment-related effects on hematology in both sexes of the 1000 mg/kg/day group consisted of statistically significant, marginal decreases in red blood cell counts, hemoglobin concentration, and hematocrit levels with a corresponding treatment-related increase in reticulocyte counts that was consistent with compensatory response to lower hemoglobin and hematocrit levels. However, there were no associated histopathological effects in the bone marrow or spleen of males and females given 1000 mg/kg/day.

CLINICAL CHEMISTRY
There was a statistically identified increase in total bile acids for males in the 1000 mg/kg/day group, which was interpreted to be treatment-related; however, this effect was of questionable toxicological significance as this finding was not observed in high-dose females and there were no associated histopathological effects in the liver or alterations in liver enzymes that would be expected in the presence of a toxicologically significant increase in bile acids.

URINALYSIS
Males given 1000 mg/kg/day had a higher incidence of acidic urine (pH 6.5) as compared to the controls (4/5 given 1000 mg/kg/day versus 0/5 controls). The change in the urine pH was interpreted to be treatment related, possibly due to excretion of the test material and/or its metabolites in the urine. Urine samples from some of the females in all treatment-groups were noted to be acidic (pH 6.5 or 6.0), however, due to absence of a clear dose-response relationship, it was interpreted to be unrelated to treatment.

ORGAN WEIGHTS
Males and females given 1000 mg/kg/day had statistically significant, treatment-related increases in relative kidney weights compared to controls. The exact reason(s) for the increased relative kidney weights are not clear, although kidneys of males and females at this dose level had a marginal treatment-related increase in the severity of tubular mineralization, relative to controls.
Females in the 1000 mg/kg/day group had a statistically higher mean relative spleen weight compared to controls which was interpreted to be treatment-related as this value was outside recent historical control range; however, this change was not associated with any treatment-related histopathological effect.
There were statistically identified decreases in absolute (males at 100 mg/kg/day; females at 300 and 1000 mg/kg/day) and relative (females at 300 and 1000 mg/kg/day) heart weights; however, these alterations were unrelated to treatment as these values were within recent historical control ranges, and there was a lack of a dose-response relationship. Moreover, there were no histopathological correlates to the lower heart weights at 1000 mg/kg/day.

GROSS PATHOLOGY
Treatment-related gross observations at necropsy were limited to the stomach and consisted of a slight thickening of the limiting ridge (a fold of the stomach mucosa that demarcates the border between the forestomach and the glandular stomach) in males and females given 1000 mg/kg/day as compared to the respective controls. All other gross findings were interpreted to be spontaneous changes unassociated with exposure to the test material.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related histopathological changes were limited to the stomach and kidneys of males and females given 300 or 1000 mg/kg/day. Treatment-related stomach effects were noted in the glandular mucosa as well as at the limiting ridge of the non-glandular mucosa of the forestomach. Males and females given 300 or 1000 mg/kg/day had a treatment-related, dose-dependent, very slight or slight hyperplasia of the neck mucous cells of the glandular mucosa.
There were no treatment-related histopathological changes in the stomachs of males and females given 100 mg/kg/day.
In the kidneys of males and females given 300 or 1000 mg/kg/day, there was a marginal treatment-related increase in the degree of background mineralization of medullary tubules as compared to that observed in the controls.
There were no treatment-related histopathological changes in the kidneys of males and females given 100 mg/kg/day.

HISTORICAL CONTROL DATA (if applicable)
Sex Males
Historical Control@
Red Blood Cell Count (E6/ul) 8.68-9.07
Hemoglobin Concentration (g/dl) 15.7-17.5
Hematocrit (%) 48.0-50.4
Reticulocytes (109/l) 143.3-180.4
Sex Females
Historical Control@
Red Blood Cell Count (E6/ul) 8.67-8.73
Hemoglobin Concentration (g/dl) 16.0-17.0
Hematocrit (%) 47.8-48.8
Reticulocytes (109/l) 138.6-165.1


Sex Males
Historical Control@
Total Bile Acids (umol/l) 3.70-17.18
Total protein (g/dl) 6.6-6.8
Albumin (g/dl) 4.5-4.6
Sex Females
Historical Control@
Total protein (g/dl) 6.4-6.6
Albumin (g/dl) 4.5-4.6

Sex Males
Historical Control@
Final Body wt (g) 198.5-212.2
Heart (g) 0.646-0.694
Kidneys (g/100) 0.663-0.729
Sex Females
Historical Control@
Final Body wt (g) 129.6-140.3
Heart (g) 0.472-0.522
Heart (g/100) 0.336-0.403
Kidneys (g/100) 0.790-0.810
Spleen (g/100) 0.267-0.273

@Historical control data obtained from four 28-day gavage studies conducted in this laboratory in 2012
using PG as the vehicle.


Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified

Table 1: Clinical Observations Summary

 MALES
 DOSE     0  100  300 1000
 Within Normal limits        
 DAYS  1 -6  5/5  5/5  5/5  5/5
 DAY  7  5/5  4/5  5/5  5/5
 DAYS  8 -10  5/5  4/5  4/5  5/5
 DAYS  11 -22  5/5  4/5  5/5  5/5
 DAYS  23 -27  5/5  4/5  4/5  5/5
 DAY  28  3/5  4/5  4/5  5/5
 Injury, Apparent Mechanical        
 DAYS  8 -10  0/5  0/5  1/5  0/5
 DAY  28  1/5  0/5  0/5  0/5
 Posture, Head-Tilt        
 DAYS  7 -28  0/5  1/5  0/5  0/5
 Skin/Fur/Mucous Membranes, Excessive Hairloss        
 DAYS  23 -27  0/5  0/5  1/5  0/5
 DAY  28  1/5  0/5  1/5  0/5
                FEMALES
 DOSE     0  100  300  1000
 Within Normal Limits           
 DAYS  1 -22  5/5  5/5  5/5  5/5
 DAYS  23 -28  5/5  5/5  5/5  4/5
 Skin/Fur/Mucous Membranes, Excessive Hairloss        
 DAYS  23 -28  0/5  0/5  0/5  1/5

Table 2: Hematology Summary

 MALES
 Dose(mkd)    WBC(E3/ul)  RBC(E6/ul)  HGB(g/dl)  HCT(pcnt) MCV(fl)   MCH(pg)  MCHC(g/dl)  PLT(E3/ul)  RET(E9/l)
 0  Mean  6.48  8.66  15.8  48.7  56.2  18.3  32.5  804  190.3
   S.D.  1.36  0.14  0.2  0.6  0.9  0.2  0.5  91  17.8
   N=  5  5  5  5  5  5  5  5  5
 100  Mean  7.10  8.70  15.8  48.2  55.4  18.1  32.8  736  172.6
   S.D.  1.29  0.17  0.3  1.1  1.1  0.3  0.4  67  23.6
   N=  5  5  5  5  5  5  5  5  5
 300  Mean  6.82  8.67  15.8  48.4  55.9  18.2  32.5  834  190.8
   S.D.  1.25  0.29  0.3  1.4  0.6  0.2  0.4  37  5.9
   N=  5  5  5  5  5  5  5  5  5
 1000  Mean  7.29  8.48  15.6  48.1  56.6  18.4  32.5  784  231.8
   S.D.  1.28  0.17  0.4  0.7  0.5  0.4  0.7  78  40.1
   N=  5  5  5  5  5  5  5  5  5
                            FEMALES  
 Dose(mkd)    WBC(E3/ul)  RBC(E6/ul)  HGB(g/dl)  HCT(pcnt)  MCV(fl)  MCH(pg)  MCHC(g/dl)  PLT(E3/ul)  RET(E9/l)
 0  Mean  8.32  8.85  16.5  48.4  54.7  18.7  34.1  711  141.7
   S.D.  1.72  0.11  0.2  1.1  0.7  0.1  0.4  117  5.3
   N=  5  5  5  5  5  5  5  5  5
 100  Mean  7.81  8.67  16.3  47.9  55.3  18.8  34.1  795  140.2
   S.D.  1.24  0.10  0.4  0.7  0.5  0.3  0.8  96  22.6
   N=  5  5  5  5  5  5  5  5  5
 300  Mean  8.18  8.94  16.4  49.3  55.1  18.3  33.2  814  141.8
   S.D  1.51  0.38  0.5  2.1  0.1  0.4  0.6  54  10.8
   N=  5  5  5  5  5  5  5  5  5
 1000  Mean  7.94  8.24  15.6  46.0  55.8  19.0  34.0  844  156.8
   S.D.  1.29  0.23  0.2  1.4  0.3  0.3  0.6  87  9.7

WBC=TOTAL LEUKOCYTE COUNT, RBC=ERYTHROCYTE COUNT, HGB=HEMOGLOBIN CONCENTRATION,

HCT=HEMATOCRIT, MCV=MEAN CORPUSCULAR VOLUME, MCH=MEAN CORPUSCULAR HEMOGLOBIN,

MCHC=MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION, PLT=PLATELET COUNT, RET=RETICULOCYTE COUNT

Table 3: Clinical Chemistry Summary

 MALES
 Dose(mkd)    ALT(u/l)  ALP(u/l)  AST(u/l)  GGT(u/l)  TBIL(mg/dl)  TBA(umol/l)  TP(g/dl)  ALB(g/dl)  GLOB(g/dl)  A/GRatio  CHOL(mg/dl)  TRIG(mg/dl)
 0  Mean  39  164  84  1.5  0.05  4.51  6.7  4.6  2.1  2.2  62  61
   S.D.  4  9  6  0.0  0.00  1.98  0.2  0.2  0.1  0.1  3  11
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 100  Mean  38  156  81  1.5  0.05  8.64  6.6  4.5  2.1  2.2  60  49
   S.D.  2  16  6  0.0  0.00  8.78  0.2  0.1  0.1  0.1  7  9
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 300  Mean  39  166  90  1.5  0.05  6.66  6.6  4.5  2.0  2.2  60  52
   S.D.  4  9  13  0.0  0.00  2.94  0.2  0.2  0.1  0.2  4  9
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 1000  Mean  41  166  87  1.5  0.05  21.34  6.2  4.3  1.9  2.3  55  62
   S.D.  6  8  12  0.0  0.00  12.28  0.1  0.1  0.1  0.1  5  8
   N=  5  5  5  5  5  5  5  5  5  5  5  5
GGT LOWEST DETECTION LIMIT IS 3.0. INDIVIDUAL VALUES <3.0 WERE REPORTED AS 1.5 (ONE-HALF THE DETECTION LIMIT) FORSTATISTICAL ANALYSIS.TBIL LOWEST DETECTION LIMIT IS 0.10. INDIVIDUAL VALUES <0.10 WERE REPORTED AS 0.05 (ONE-HALF THE DETECTION LIMIT) FORSTATISTICAL ANALYSIS.ALT=ALANINE AMINOTRANSFERASE, ALP=ALKALINE PHOSPHATASE, AST=ASPARTATE AMINOTRANSFERASE,GGT=GAMMA GLUTAMYL TRANSPEPTIDASE TBIL=TOTAL BILIRUBIN, TBA = TOTAL BILE ACIDS, TP=TOTAL PROTEIN,ALB=ALBUMIN, GLOB=GLOBULIN, A/G=RATIO OF ALBUMIN/GLOBULIN, CHOL=CHOLESTEROL, TRIG=TRIGLYCERIDES  
 
MALES (cont.)
 Dose(mkd)    UN(mg/dl)  CREA(mg/dl)  GLUC(mg/dl)  NA(mmol/l)  K(mmol/l)  CL(mmol/l)  CA(mg/dl)  PHOS(mg/dl)  
     
     
 0  Mean  19  0.3  125  149  5.8  97  12.8  12.9        
   S.D.  2  0.0  12  1  0.3  1  0.2  1.4        
   N=  5  5  5  5  5  5  5  5        
 100  Mean  17  0.2  130  149  5.9  97  12.8  12.9        
   S.D.  1  0.1  23  1  0.5  1  0.5  0.8        
   N=  5  5  5  5  5  5  5  5        
 300  Mean  18  0.3  122  148  5.9  97  12.8  12.8        
   S.D.  1  0.1  18  1  0.2  2  0.3  0.7        
   N=  5  5  5  5  5  5  5  5        
 1000  Mean  17  0.2  131  148  6.4  96  12.9  13.8        
   S.D.  2  0.1  24  1  0.4  1  0.5  1.0        
   N=  5  5  5  5  5  5  5  5        
UN=UREA NITROGEN, CREA=CREATININE, GLUC=GLUCOSE, NA=SODIUM, K=POTASSIUM, CL=CHLORIDE,CA=CALCIUM, PHOS=PHOSPHORUS
 
FEMALES
 Dose(mkd)    ALT(u/l)  ALP(u/l)  AST(u/l)  GGT(u/l)  TBIL(mg/dl)  TBA(umol/l)  TP(g/dl)  ALB(g/dl)  GLOB(g/dl)  A/GRatio  CHOL(mg/dl)  TRIG(mg/dl)
 0  Mean  33  134  90  1.5  0.05  16.32  6.3  4.5  1.9  2.4  74  51
   S.D  3  5  5  0.0  0.00  17.98  0.3  0.1  0.2  0.2  3  8
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 100  Mean  29  131  82  3.4  0.05  7.92  6.1  4.4  1.7  2.5  68  49
   S.D.  2  6  8  4.3  0.00  3.77  0.2  0.2  0.1  0.2  5  10
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 300  Mean  33  136  87  1.5  0.05  5.25  6.4  4.5  1.9  2.4  72  57
   S.D.  5  16  11  0.0  0.00  1.58  0.4  0.2  0.2  0.2  8  7
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 1000  Mean  30  128  84  1.5  0.05  12.31  6.0  4.3  1.7  2.5  68  45
   S.D.  1  8  3  0.0  0.00  10.59  0.2  0.1  0.1  0.2  9  4
   N=  5  5  5  5  5  5  5  5  5  5  5  5
 GGT LOWEST DETECTION LIMIT IS 3.0. INDIVIDUAL VALUES <3.0 WERE REPORTED AS 1.5 (ONE-HALF THE DETECTION LIMIT) FORSTATISTICAL ANALYSIS.TBIL LOWEST DETECTION LIMIT IS 0.10. INDIVIDUAL VALUES <0.10 WERE REPORTED AS 0.05 (ONE-HALF THE DETECTION LIMIT) FORSTATISTICAL ANALYSIS.ALT=ALANINE AMINOTRANSFERASE, ALP=ALKALINE PHOSPHATASE, AST=ASPARTATE AMINOTRANSFERASE,GGT=GAMMA GLUTAMYL TRANSPEPTIDASE TBIL=TOTAL BILIRUBIN, TBA = TOTAL BILE ACIDS, TP=TOTAL PROTEIN,ALB=ALBUMIN, GLOB=GLOBULIN, A/G=RATIO OF ALBUMIN/GLOBULIN, CHOL=CHOLESTEROL, TRIG=TRIGLYCERIDES
 
FEMALES (cont.)
 Dose(mkd)    UN(mg/dl)  CREA(mg/dl)  GLUC(mg/dl)  NA(mmol/l)  K(mmol/l)  CL(mmol/l)  CA(mg/dl)  PHOS(mg/dl)        
 0  Mean  15  0.3  92  149  6.6  97  13.0  14.6        
   S.D.  1  0.1  14  3  0.5  2  0.4  1.5        
   N=  5  5  5  5  5  5  5  5        
 100  Mean  21  0.4  98  148  6.6  98  12.4  14.0        
   S.D.  10  0.5  10  1  0.9  2  0.3  2.4        
   N=  5  5  5  5  5  5  5  5        
 300  Mean  16  0.2  108  151  6.4  99  12.9  13.4        
   S.D.  1  0.1  13  7  0.5  5  0.6  0.6        
   N=  5  5  5  5  5  5  5  5        
 1000  Mean  19  0.2  111  148  6.4  98  12.5  13.6        
   S.D.  1  0.0  9  1  0.6  0  0.4  1.2        
   N=  5  5  5  5  5  5  5  5        

UN=UREA NITROGEN, CREA=CREATININE, GLUC=GLUCOSE, NA=SODIUM, K=POTASSIUM, CL=CHLORIDE,

CA=CALCIUM, PHOS=PHOSPHORUS

Table 4: Urinalysis Summary  

FEMALES 
 Dose(mkd)    UrineVolume(ml)  SpecificGravity  Color  Appear  pH  Protein  Glucose  Ketones  Bilirubin  Blood  Urobilinogen(eu/dl)
 0  Mean  2.5  1.069  yellow (4)  clear (4)  7.0 (1)  + (3)  Neg  TRC (4)  + (4)  Neg (5)  1.0 (1)
   S.D.  0.8  0.009  DK. YE (1)  cloudy (1)  7.5 (1)  ++ (2)    + (1)  ++ (1)    2.0 (3)
   N=  5  5      8.5 (3)            4.0 (1)
 100  Mean  4.1  1.051  yellow (5)  clear (5)  6.5 (3)  Neg (1)  Neg (5)  Neg (2)  Neg (2)  Neg (5)  0.2 (1)
   S.D.  2.2  0.014      7.0 (1)  TRC (3)    TRC (3)  + (3)    1.0 (4)
   N=  5  5      7.5 (1)  + (1)          
 300  Mean  2.7  1.064  yellow (5)  clear (5)  6.0 (1)  TRC (1)  Neg (5)  Neg (1)  Neg (1)  Neg (5)  1.0 (3)
   S.D.  0.6  0.012      7.0 (2)  + (4)    TRC (4)  + (3)    2.0 (2)
   N=  5  5      7.5 (2)        ++ (1)    
 1000  Mean  4.5  1.051  yellow (5)  clear (4)  6.0 (2)  Neg (1)  Neg (5)  Neg (5)  Neg (2)  Neg (5)  0.2 (1)
   S.D.  2.6  0.014    SL CL (1)  6.5 (2)  TRC (3)      + (3)    1.0 (4)
   N=  5  5      7.0 (1)  + (1)          
 URINE VOLUME AND SPECIFIC GRAVITY VALUES ARE MEAN AND S.D. FOR THE SPECIFIED NUMBER (N) OF ANIMALS.ALL OTHER DATA TABULATED AS NUMBER OF ANIMALS (N) WITH THE STATED VALUE.DK.YE=DARK YELLOW SL CL=SLIGHTLY CLOUDY EU/DL=EHRLICH UNITS/DECILITERNEG=NEGATIVE TRC=TRACE +=SMALL ++=MODERATE +++=LARGE
 
MALES
 Dose(mkd)    UrineVolume(ml)  SpecificGravity  Color  Appear  pH  Protien  Glucose  Ketones  Bilirubin  Blood  Urobilinogen(eu/dl)
 0  Mean  5.4  1.048  yellow (5)  clear (5)  7.0 (1) + (5)  Neg (5)  TRC (4)  Neg (4)  Neg (5)  1.0 (5)
   S.D.  1.0  0.009      7.5 (1)      + (1)  + (1)    
   N=  5  5      8.0 (3)            
 100  Mean  4.1  1.056  yellow (5)  clear (4)  7.5 (3)  + (5)  Neg (5)  TRC (2)  Neg (2)  Neg (5)  1.0 (4)
   S.D.  1.0  0.006    SL CL (1)  8.0 (2)      + (3)  + (3)    2.0 (1)
   N=  5  5                  
 300  Mean  4.3  1.055  yellow (5)  clear (3)  7.0 (4)  + (4)  Neg (5)  TRC (5)  Neg (4)  Neg (5)  1.0 (5)
   S.D.  0.8  0.008    SL CL (2)  7.5 (1)  ++ (1)      + (1)    
   N=  5  5                  
 1000  Mean  6.1  1.050  yellow (5)  clear (5)  6.5 (4)  + (5)  Neg (5)  Neg (4)  Neg (5)  Neg (5)  1.0 (5)
   S.D.  1.3  0.008      7.0 (1)      TRC (1)      
   N=  5  5                  
URINE VOLUME AND SPECIFIC GRAVITY VALUES ARE MEAN AND S.D. FOR THE SPECIFIED NUMBER (N) OF ANIMALS. ALL OTHER DATA TABULATED AS NUMBER OF ANIMALS (N) WITH THE STATED VALUE. DK.YE=DARK YELLOW SL CL=SLIGHTLY CLOUDY EU/DL=EHRLICH UNITS/DECILITER NEG=NEGATIVE TRC=TRACE +=SMALL ++=MODERATE +++=LARGE
 
Table 5: Gross Pathology Observations
   MALES            FEMALES        

   0  100  300  1000  0  100  300  1000
   MKD  MKD  MKD  MKD  MKD  MKD  MKD  MKD
 Number of Animals on Study  5  5  5  5  5  5  5  5
 Number of Animals Completed  (5)  (5)  (5)  (5)  (5)  (5)  (5) (5)
 STOMACH                
  Submitted  (5)  (5)  (5)  (5)  (5)  (5)  (5)  (5)
  No Visible Lesions  5  5  5  0  5  5  5  0
  Thickened; Limiting Ridge  0  0  0  5  0  0  0  5

Table 6: Histopathological Observations

   MALES           FEMALES         
   0  100  300  1000  0  100  300  1000
 Number of Animals on Study  5  5  5  5  5  5  5  5
 Number of Animals Completed  (5)  (5)  (5)  (5)  (5)  (5)  (5)  (5)
 KIDNEYS                
  Examined  (5)  (5)  (5)  (5)  (5)  (5)  (5)  (5)
  Within Normal Limits  0  0  0  0  0  0  0  0
  Degeneration; Tubule; Focal  (1)  (1)  (1)  (0)  (0)  (0)  (0)  (0)
  very slight  1  1  1  0  0  0  0  0
  Degeneration; Tubule; Mulitfocal  (1)  (0)  (2)  (0)  (0)  (0)  (0)  (2)
  very slight  1  0  2  0  0  0  0  2
  Dilatation; Tubule; Medulla; Focal  (0)  (0)  (1)  (0)  (0)  (2)  (0)  (0)
  very slight  0  0  1  0  0  2  0  0
  Inflammation; Subacute to chronic; Interstitium; Focal  (0)  (0)  (0)  (1)  (0)  (1)  (0)  (0)
  very slight  0  0  0  1  0  1  0  0
  Inflammation; Subacute to chronic; Interstitium; Mulitfocal  (0)  (0)  (0)  (0)  (0)  (0)  (0)  (1)
  very slight  0  0  0  0  0  0  0  1
  Mineralization; Tubule; Medulla; Multifocal  (5)  (5)  (5)  (5)  (5)  (5)  (5)  (5)
  very slight  5  5  3  0  5  5  4  1
  slight  0  0  2  5  0  0  1  4
 STOMACH                
 Examined  (5)  (5)  (5)  (5)  (5)  (5)  (5)  (5)
 Within Normal Limits  5  5  0  0  4  5  2  0
 Dilatation; gland; glandular mucosa; focal  (0)  (0)  (0)  (1)  (0)  (0)  (0)  (0)
 very slight  0  0  0  1  0  0  0  0
 Edema; limiting ridge; nonglandular mucosa  (0)  (0)  (3)  (5)  (1)  (0)  (0)  (3)
 very slight  0  0  3  2  0  0  0  2
 slight  0  0  3  1  0  0  1
 Hyperplasia; neck mucous cell; glandular mucosa  (0)  (0)  (5)  (5)  (0)  (0)  (3)  (5)
 very slight  0  0  5  0  0  0  3  0
 slight  0  0  0  5  0  0  0  5
 Hyperplasia; epithelial; limiting ridge; nonglandular mucosa  (0)  (0)  (2)  (5)  (0)  (0)  (0)  (5)
 very slight  0  0  2  5  0  0  0  3
 slight  0  0  0  0  0  0  0  2
 Inflammation; subacute; glandular mucosa; multifocal  (0)  (0)  (0)  (3)  (0)  (0)  (0)  (2)
 very slight  0  0  0  3  0  0  0  2
 Inflammation; subacute; limiting ridge; nonglandular mucosa  (0)  (0)  (2)  (5)  (1)  (0)  (0)  (3)
 very slight  0  0  2  4  1  0  0  2
 slight  0  0  0  1  0  0  0  1

Table 7:Text Table 5. Selected Final Body and Organ Weights

 Sex  MALES         
 Dose (mg/kg/day)  0  100  300  1000
 Final Body wt (g)  216.4  208.1  211.4  210.2
 Heart (g)  0.737  0.664a  0.682  0.732
 Kidneys (g/100)  0.715  0.724  0.751  0.775*
 Sex

 FEMALES         

 Dose (mg/kg/day)  0  100  300  1000
 Final Body wt (g)  134.5  135.4  136.0  131.9
 Heart (g)  0.519  0.505  0.487a  0.483a
 Heart (g/100)  0.386  0.373  0.358a  0.366a
 Kidneys (g/100)  0.793  0.777  0.789  0.833*
 Spleen (g/100)  0.277  0.273  0.276  0.298a

*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 judged to be treatment related.

Conclusions:
Under the conditions of this study, based on histopathological changes in the stomach and kidney of males and females given 300 and 1000 mg/kg/day, the no-observed-effect level (NOEL) for HEMA phosphate in F344/DuCrl rats of either sex was 100 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the potential toxicity of reaction product of 2 -hydroxyethyl methacrylate (HEMA) and polyphosphoric acid (PPA), hereafter referred to as HEMA phosphate, in rats following oral gavage administration for 28 days. Five male and five female F344/DuCrl rats per group were administered 0, 100, 300, or 1000 milligrams HEMA phosphate per kilogram body weight per day (mg/kg/day, mkd) in propylene glycol (PG) via gavage. Parameters evaluated were daily cage-side and clinical observations, weekly detailed clinical observations, ophthalmic examinations, body weights/body weight gains, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, gross and histopathological examinations.

There were no treatment-related effects in clinical signs, body weights, feed consumption, ophthalmic examinations, or prothrombin time for any treatment group. Males and females given 1000 mg/kg/day had marginal treatment-related decreases in hematocrit, red blood cell counts and hemoglobin levels along with associated increases in reticulocyte counts. In addition there were slight treatment-related decreases in total protein and albumin levels in these dose groups. Serum total bile acid levels in males given 1000 mg/kg/day were higher than the controls and statistically identified; however, this was interpreted to be of questionable toxicological significance due to lack of any associated treatment-related liver histopathological effects or change in liver enzymes. Males given 1000 mg/kg/day had a treatment-related higher incidence of lower urine pH (6.5) as compared to the controls, possibly due to the excretion of the test material and/or its metabolites in the urine.

There were no treatment-related effects on hematology, clinical chemistry or urinalysis parameters for males or females in the 100 or 300 mg/kg/day dose groups. Treatment-related organ weight effects were limited to the 1000 mg/kg/day group and consisted of increased relative kidney weights in both sexes, and an increased relative spleen weight in females only. There were no treatment-related organ weight effects in either sex in the 100 or 300 mg/kg/day dose groups.

Treatment-related gross pathological observations were limited to the stomach of males and females given 1000 mg/kg/day and consisted of a slight thickening of the limiting ridge. Treatment-related histopathological effects were limited to the stomach and kidneys of males and females given 300 or 1000 mg/kg/day. In the stomach, treatment-related histopathological changes consisted of very slight or slight hyperplasia of the neck mucous cells of the glandular mucosa, for males and females in the 300 or 1000 mg/kg/day groups. A very slight subacute multifocal inflammation was also observed in the glandular mucosa of some of the males and females given 1000 mg/kg/day. Males and females given 1000 mg/kg/day and males given 300 or 1000 mg/kg/day had very slight or slight epithelial hyperplasia of the limiting ridge which was also accompanied by very slight or slight subacute inflammation and a very slight edema in the lamina propria underlying the epithelium. Histopathological changes in the stomach were interpreted to be due to localized, point of contact irritant effects due to the repeated oral gavage of the test material. In the kidneys of males and females given 300 or 1000 mg/kg/day, there was a marginal treatment-related increase in the degree of background mineralization of medullary tubules as compared to that observed in the controls.

There were no treatment-related gross or histopathological alterations for males and females given 100 mg/kg/day.

Under the conditions of this study, based on histopathological changes in the stomach and kidney of males and females given 300 and 1000 mg/kg/day, the no-observed-effect level (NOEL) for HEMA phosphate in F344/DuCrl rats of either sex was 100 mg/kg/day.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 24, 2015-November 24,2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
other: JMAFF. Testing Guidelines for Toxicology Studies, 2000 (90-Day Repeated Oral Toxicity Studies, 2-1-9)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Remarks:
F344/DuCrl
Details on species / strain selection:
F344/DuCrl rats were selected because of their general acceptance and suitability for toxicity testing, previous toxicity studies on HEMA phosphate have used this strain, availability of historical background data, and the reliability of the commercial supplier.
Sex:
male/female
Details on test animals and environmental conditions:
Supplier and Location: Charles River (Kingston, New York)
Age at Study Start: Approximately 7 weeks
Physical and Acclimation: Upon arrival, the animals were housed two to three per cage in stainless steel cages. Cages had solid floors with corncob bedding and paper twists and/or nylon bones for enrichment. Animals were housed in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International) for approximately one week prior to the start of 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.
Housing: After assignment, animals were housed two per cage in stainless steel cages. Cages had solid floors with ground corncob bedding and paper twists and/or nylon bones for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were targeted in the animal room, however temporary excursions from these environmental conditions may have occurred on an infrequent basis; 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.)
Enrichment: Enrichment for rats included the use of ground corn cob bedding, paper twists and/or nylon bones, and open areas on the cage sides for visualization of other rats.
Feed and Water: Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. 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. Copies of these analyses were maintained in the study file.
Route of administration:
oral: gavage
Details on route of administration:
A possible route of human exposure to the test material would be via accidental ingestion. Thus, oral administration of the test material to rats via oral gavage represented an appropriate means of exposure.
Vehicle:
propylene glycol
Details on oral exposure:
All dosing solutions were prepared by mixing the test material in PG (lot #P4347, Sigma-Aldrich, St. Louis, Missouri) at concentrations of 5, 16.7, or 50 mg/mL and administered at a dose volume of 6 mL/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted at least weekly based on individual body weights. The control rats were dosed with PG at 6 mL/kg body weight. Dose solutions were prepared periodically throughout the study based on stability data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Confirmation and Homogeneity: Dose confirmation analyses of all dose solutions were determined pre-exposure, near the middle, and end of the study. The homogeneity of the low-dose and the high-dose test solutions was determined concurrent with dose confirmation. The method used for analyzing the test material in PG was high performance liquid chromatography with ultraviolet detection (HPLC/UV) (Malowinski, 2013).The overall mean concentrations ranged from 94.3 to 104.7% of targeted concentrations indicating acceptable concentrations of HEMA phosphate. The overall mean relative standard deviations were between 0.2 and 1.4% indicating that the test solutions were homogeneously mixed.
Stability: HEMA phosphate was stable for at least 25 days in PG at concentrations ranging from 0.025 to 250 mg/mL (Malowinski, 2013) using HPLC/UV for analyses. This established stability concentration range and duration spanned those used in this study. The NTM stability of HEMA Phosphate (Lot 10236921) was evaluated by comparing the % of target concentration (calculation of standard refit data) obtained from the initial calibration standards to the percent of target concentration data obtained from subsequent calibration standards. HEMA Phosphate test material used was sufficiently stable to meet the needs of this 90-day study.
Solubility: Solubility was confirmed in PG at concentrations up to 500 mg/mL prior to the start of dosing (data maintained in the study file).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 7days/week
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Randomization and Identification: Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

Dose Levels and Justification: The dose levels selected were based on the results of the previous 28-day study that showed a NOEL of 100 mg/kg/day based on histopathological changes in the stomach and kidney of males and females given 300 and 1000 mg/kg/day. The high dose of 300 mg/kg/day was expected to induce point of contact effects, such as stomach irritation. 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:
Daily Observations: A cage-side examination was conducted on all animals at least once a day upon arrival, at approximately the same time each day (usually in the morning). 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 have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity or color. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Clinical Observations: Clinical observations were conducted daily approximately one hour after dosing and included a careful 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.
Detailed Clinical Observations: Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout 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 near the end of the treatment period
for all animals.
Sensory Evaluation: The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box.
Rectal Temperature: Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37°C before, during, and after the study. The instrument was recalibrated if the temperature recordings differed from the reference thermometer by more than ± 0.5°C.
Grip Performance: Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forelegs on a plastic bench and the hindfeet were set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken. An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet were suspended approximately 10 cm above the plastic platform.
Motor Activity: A commercially-available automated test system (MotorMonitor, Kinder Scientific, Inc., Poway, CA) was used for motor activity (MA) data collection. Each animal was tested individually in one of 24 available square acrylic enclosures (approximately 22” X 22”) which contained a grid of infra-red beams (32 total beams, 16x and 16y) near the floor of the enclosure. The MA test room was maintained under white noise conditions (55-65 dB) and with the lighting off during testing. No entry into the MA test room was allowed during the testing period. Each test session consisted of six 10-minute intervals, totaling 60 minutes of testing per animal per test session. All beam break “activity counts” for each interval were recorded.
Ophthalmology: 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 topically 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.
Body Weights/Body Weight Gains: All rats were weighed pre-exposure and then weekly during the dosing period. Body weight gains were calculated relative to day 1.
Feed Consumption: Feed consumed was determined weekly for all animals by weighing feed containers at the start and end of a measurement cycle. Feed consumption was calculated using the following equation: Feed consumption (g/day) = (initial Wt. of feeder - final Wt. of feeder) / (# of days in measurement cycle) (# of animals in cage)
Clinical Pathology: 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 O2 at the scheduled necropsy. Blood was not obtained from animals that died or were euthanized in a moribund condition prior to their scheduled necropsy.
Hematology: Hematocrit (HCT), Hemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Neutrophils (NEUT), Lymphocytes (LYMP), Monocytes (MONO), Eosinophils (EOS), Basophils (BASO), Large Unstained Cells (LUC) which included atypical lymphocytes, large lymphocytes, plasma cells, and blasts
Platelet (PLT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC)
Manual differential WBC counts were performed as appropriate per department Standard Operating Procedures.
Coagulation: Prothrombin time (PT)
Clinical Chemistry: Enzyme Activities of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT); Concentrations of: Albumin (ALB), Albumin/Globulin Ratio (A/G) - calculated, Cholesterol (CHOL), Creatinine (CREA), Calcium (CA), Phosphorus (PHOS), Sodium (NA), Potassium (K), Chloride (CL), Globulin (GLOB) - calculated,Glucose (GLUC), Total bile acids (TBA), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), Urea nitrogen (UN)
Urinalysis: Urine samples were obtained from all surviving 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. Assays: Color, appearance, specific gravity (refractometer), and urine volume, pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen; Microscopic Examination: Urine samples were also collected from each animal by manual compression of the urinary bladder. The urine samples were pooled from each group and the microsediment was characterized microscopically.
Sacrifice and pathology:
Fasted rats were weighed, submitted alive for necropsy, anesthetized by the inhalation of isoflurane vapors and O2, and blood samples were obtained from the orbital sinus.
A complete necropsy was conducted on all animals. 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. Theeyes 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 brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, and spleen were trimmed and weighed immediately. The thyroid with the parathyroid gland(s) was trimmed and weighed following fixation and dissection from the trachea. The ratios of organ weight to terminal body weight were calculated.
Representative samples of tissues listed below were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in with the tissues. Similar necropsy procedures were followed for animals found dead or moribund, except that body weights, organ weights, and blood samples were not obtained.
Histopathology: The numbers of sections from all preserved tissues listed below were processed by standard histologic procedures from control and high-dose group animals and all animals that were sacrificed in a moribund condition. Paraffin embedded tissues were sectioned approximately six μm thick, stained with hematoxylin and eosin, and examined by a veterinary pathologist using a light microscope. Relevant gross lesions and all target tissues were microscopically examined from all animals in the low- and intermediate-dose groups.
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normaltextbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade would have been used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may be life threatening.
Statistics:
See attached description of statistics and calculations.
Clinical signs:
no effects observed
Description (incidence and severity):
On TD 58 animal #3871 in the control group had observations of sneezing, noisy upper airway, and labored mouth breathing. This animal was euthanized and necropsy revealed a gavage dosing error. There were no clinical findings or scored DCO observations that were associated with exposure to HEMA phosphate.
Mortality:
no mortality observed
Description (incidence):
All rodents survived the 90-day test period with the exception of one control male, animal #3871, which was euthanized on TD 58 due to observations of sneezing, noisy upper airway, and labored mouth breathing. Necropsy revealed a slightly viscous material (dosing vehicle) in the lung indicating a gavage dosing error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains of males in the 300 mg/kg/day group showed a treatment-related decrease of 16.3% on TD 8, compared to control; however, the decrease in percent body weight gain lessened over the course of the study. At the end of the study, the body weight gain of males in the 300 mg/kg/day group was 4.4% lower than controls. Body weight gains of males in all other groups were similar to controls. There were no treatment-related effects on body weights for males or females at any dose level. Body weights of males and females were similar to control for all dose groups throughout the duration of the study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related differences in the amount of feed consumed by males or females at any dose level throughout the duration of the study. There were a few sporadic incidences of slightly lower feed consumption measurements that were statistically identified for both males (100 mg/kg/day) and females (100 and 300 mg/kg/day). These lower values were minimal, inconsistent, and lacked a dose response, therefore, they were considered spurious and unrelated to treatment with HEMA phosphate.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Pre-exposure ophthalmological observations included cloudy cornea (one high-dose male, one control female, and one low-dose female) and persistent hyaloid artery (one control male). Prior to study termination (TD 88), ophthalmological observations included cloudy cornea (one high-dose male, one control female, four low-dose females, and one mid-dose female), incomplete dilation (one control female), attenuated blood vessels (one control male, one mid-dose male, and one mid-dose female), irregular corneal surface (one control female), hyper-reflective fundus (one mid-dose male), and periocular soiling (two low-dose females). These observations were interpreted to be unrelated to treatment due to low incidence and lack of a dose response.
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Body weight gains of males in the 300 mg/kg/day group showed a treatment-related decrease of 16.3% on TD 8, compared to control; however, the decrease in percent body weight gain lessened over the course of the study. At the end of the study, the body weight gain of males in the 300 mg/kg/day group was 4.4% lower than controls. There were no treatment-related effects on body weight gains in males from remaining dose groups or in females in any dose group.
There were no treatment-related effects in clinical signs, functional tests, body weights, feed consumption, ophthalmic, hematology, prothrombin time, clinical chemistry, or urinalysis parameters. There were no treatment-related organ weight effects, gross observations, or histopathologic observations.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Critical effects observed:
not specified
Conclusions:
Body weight gains of males in the 300 mg/kg/day group showed a treatment-related decrease of 16.3% on TD 8, compared to control; however, the decrease in percent body weight gain lessened over the course of the study. At the end of the study, the body weight gain of males in the 300 mg/kg/day group was 4.4% lower than controls. There were no treatment-related effects on body weight gains in males from remaining dose groups or in females in any dose group.

There were no treatment-related effects in clinical signs, functional tests, body weights, feed consumption, ophthalmic, hematology, prothrombin time, clinical chemistry, or urinalysis parameters. There were no treatment-related organ weight effects, gross observations, or histopathologic observations.

The no-observed-adverse effect level (NOAEL) was the targeted concentration of 300 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the potential toxicity of the reaction product of 2-hydroxyethyl methacrylate (HEMA) and polyphosphoric acid (PPA), hereafter referred to as HEMA phosphate, in rats following repeated oral gavage administration for at least 90 days.

Ten male and ten female F344/DuCrl rats per group were administered 0, 30, 100, or 300 milligrams HEMA phosphate/kilogram body weight/day (mg/kg/day) in propylene glycol (PG) via oral gavage for at least 90 days. Parameters evaluated were daily cage-side observations, daily clinical observations, weekly detailed clinical observations, ophthalmic examinations, functional tests, body weights, feed consumption, hematology, prothrombin time, urinalysis, clinical chemistry, selected organ weights, gross examination, and histopathologic examination.

Body weight gains of males in the 300 mg/kg/day group showed a treatment-related decrease of 16.3% on TD 8, compared to control; however, the decrease in percent body weight gain lessened over the course of the study. At the end of the study, the body weight gain of males in the 300 mg/kg/day group was 4.4% lower than controls.

There were no treatment-related effects on body weight gains in males from remaining dose groups or in females in any dose group. There were no treatment-related effects in clinical signs, functional tests, body weights, feed consumption, ophthalmic, hematology, prothrombin time, clinical chemistry, or urinalysis parameters. There were no treatment-related organ weight effects, gross observations, or histopathologic observations.

The no-observed-adverse effect level (NOAEL) was the targeted concentration of 300 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP/Guideline 28 day and 90 day studies

Additional information

In a 28 day study, males and females given 1000 mg/kg/day had marginal treatment-related decreases in hematocrit, red blood cell counts and hemoglobin levels along with associated increases in reticulocyte counts. In addition there were slight treatment-related decreases in total protein and albumin levels in these dose groups. Males given 1000 mg/kg/day had a treatment-related higher incidence of lower urine pH (6.5) as compared to the controls, possibly due to the excretion of the test material and/or its metabolites in the urine. Treatment-related organ weight effects were limited to the 1000 mg/kg/day group and consisted of increased relative kidney weights in both sexes, and an increased relative spleen weight in females only.

Treatment-related gross pathological observations were limited to the stomach of males and females given 1000 mg/kg/day and consisted of a slight thickening of the limiting ridge. Treatment-related histopathological effects were limited to the stomach and kidneys of males and females given 300 or 1000 mg/kg/day. In the stomach, treatment-related histopathological changes consisted of very slight or slight hyperplasia of the neck mucous cells of the glandular mucosa, for males and females in the 300 or 1000 mg/kg/day groups. A very slight subacute multifocal inflammation was also observed in the glandular mucosa of some of the males and females given 1000 mg/kg/day. Males and females given 1000 mg/kg/day and males given 300 or 1000 mg/kg/day had very slight or slight epithelial hyperplasia of the limiting ridge which was also accompanied by very slight or slight subacute inflammation and a very slight edema in the lamina propria underlying the epithelium. Histopathological changes in the stomach were interpreted to be due to localized, point of contact irritant effects due to the repeated oral gavage of the test material. In the kidneys of males and females given 300 or 1000 mg/kg/day, there was a marginal treatment-related increase in the degree of background mineralization of medullary tubules as compared to that observed in the controls.

There were no treatment-related gross or histopathological alterations for males and females given 100 mg/kg/day.

Under the conditions of this study, based on histopathological changes in the stomach and kidney of males and females given 300 and 1000 mg/kg/day, the no-observed-effect level (NOEL) for HEMA phosphate in F344/DuCrl rats of either sex was 100 mg/kg/day.

In a 90 day study, male and female rats were given 30, 100, and 300 mg/kg/day HEMA phosphate daily. Body weight gains of males in the 300 mg/kg/day group showed a treatment-related decrease of 16.3% on test day 8 compared to control; however, the decrease in percent body weight lessened over the course of the study. At the end of the study, the body weight gain of males in the 300 mg/kg/day group was 4.4% lower than controls. There were no treatment-related effects on body weight gains in males from remaining dose groups or in females in any dose group.

There were no treatment-related effects in clinical signs, functional tests, body weights, feed consumption, ophthalmic, hematology, prothrombin time, clinical chemistry, or urinalysis parameters. There were no treatment-related organ weight effects, gross observations, or histopathologic observations.

Under the conditions of this study, the no-observed-adverse effect level (NOAEL) was the targeted concentration of 300 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Because the 28 day study provided a more conservative assessment of the oral repeated dose toxicity for HEMA phosphate than the 90 day study, the reference dose was selected based on the 28 day study and the LOAEL is considered 300 mg/kg/day.


Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification

In a 28 day study in male and female F344/DuCrl rats, a LOAEL of 300 mg/kg/day was determined based on minimal histopathological changes in the stomach and kidney of males and females given 300 and 1000 mg/kg/day. In a 90 day study, male and female F344/DuCrl rats received 30, 100, or 300 mg/kg/day HEMA phosphate by oral gavage; no changes were observed in the stomach or kidneys of males or females at any dose level and the NOAEL was the high dose of 300 mg/kg/day. Based on the weight of evidence, classification for HEMA phosphate for specific target organ toxicity - repeat exposure is not warranted.