2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, phosphate

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, phosphate

EC Number:

258-053-2

EC Name:

2-Propenoic acid, 2-methyl-, 2-hydroxyethyl ester, phosphate

Cas Number:

52628-03-2

Molecular formula:

C6 H10 O3 . x H3 O4 P

IUPAC Name:

2-(methacryloyloxy)ethyl phosphate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): HEMA phosphate
- Composition of test material, percentage of components: the sum of six major components PEM-dimer, phosphoethyl methacrylate (PEM), phospho-di(ethyl methacrylate) (PEM diester), methyl methacrylate (MMA), HEMA dimer, and ethylene glycol dimethacrylate (EGDMA)
- Lot/batch No.: A022C73001

Test animals

Species:

rat

Strain:

other: F344/DuCrl

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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	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.

Applicant's summary and conclusion

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.