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

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Prenatal Developmental Toxicity study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

February 14, 2016 - March 9, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study information provided to support waiving of reproductive toxicity studies on the basis of a lack of adverse effects observed in reproductive tissues and organs

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 414

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Specific details on test material used for the study:

Purity 91.0%

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

female

Details on test animals or test system and environmental conditions:

Supplier and Location: Charles River (Raleigh, North Carolina)
Age and Weight at Study Start: Sexually mature adult, weighing approximately 200-250 g
Physical and Acclimation: 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. The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least four days prior to the start of dosing.
Housing: After assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated
lixit valve-type watering system. 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 and open areas on the cage sides for visualization of other rats. In addition, the cage contained paper nesting material.
Feed and Water: Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed was provided ad libitum. Municipal water was provided ad libitum with the exception of a single day. On March 08, 2106, water was not provided ad libitum as a result of a planned shutdown of the water supply. The animals were disconnected from the water supply for approximately two hours. This deviation did not negatively impact the quality or integrity of the study since the shutdown period was minimal and there was likely residual water in the lines so
animals were likely not without water. In addition, all animals appeared normal at the subsequent cage-side observation. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study.
Test material administration began on February 14, 2016 and the last group of rats was necropsied on March 9, 2016.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

All dosing solutions were prepared by mixing the test material in propylene glycol (PG, Lot MKBV7805 V, Sigma-Aldrich, St. Louis, Missouri) at concentrations of 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. The control rats were dosed with PG at 6 mL/kg body weight. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. Due to the length of the dosing period, dose solutions were prepared periodically throughout the study.

Details on mating procedure:

Sexually mature virgin females were naturally mated with males of the same strain (one male:one female) at the supplier. Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study record. Rats arrived in our laboratory on GD 1 or 2.

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 h
omogeneously 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:

gestation day 6 through 20

Frequency of treatment:

Daily

Details on study schedule:

Necropsy was performed on gestation day 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were stratified by GD 0 body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform mean group weights and standard deviations at the start of the study.
Dose levels for this study were selected on the basis of the developmental toxicity probe study. The high dose of 1000 mg/kg/day represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study) and was expected to induce signs of maternal toxicity, including changes in hematologic parameters indicative of anemia, and point of contact irritation in the stomach. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high dose group rats

Examinations

Parental animals: Observations and examinations:

A cage-side examination was conducted twice daily, approximately at the same time each day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that are 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 thatcould have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Clinical observations were conducted on all animals at least once daily. Animals were observed approximately one hour after dosing. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Body weights were recorded on GD 0 by the supplier and daily from GD 6-21. Statistical analysis of body weights was performed using data collected on GD 0, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

Feed consumption was recorded and statistically analyzed for all animals every three days from GD 3-21 by weighing feed containers at the start and end of a measurement cycle and consumption was calculated using the following equation: Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / (# of days in measurement cycle).

Animals were not fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy on GD 21.
Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright- Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.
Hematology parameters examined: 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 include, 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)

On GD 21, all surviving females (not fasted) were sedated with a mixture of isoflurane vapors and medical oxygen, and a blood sample was obtained from the orbital sinus. The animals were euthanized by carbon dioxide inhalation followed by cervical dislocation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies were counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification. The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The liver, kidneys, and spleen were dissected from the carcass and were incised. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal body weight were calculated. Representative portions of liver, kidneys, stomach, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of the liver, kidneys, stomach and gross lesions were not conducted.
A detailed examination of the reproductive tract was performed, and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicates a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted. The uteri of females lacking visible implantations were stained with a 10% aqueous solution of sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Litter observations:

The sex and body weight of all viable fetuses were recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods based on Trueman et al. (1999) and Zablotny (2002). A thorough evaluation of the fetal skeleton was
conducted on the remaining fetuses not selected for visceral examination. However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination provided more meaningful data about a suspected abnormality.

All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment group assignment.

Statistics:

Maternal body weights, maternal body weight gains, appropriate hematologic data, organ weights (absolute and relative with the exception of only absolute weight for gravid uterus), fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for homogeneity of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.

Frequency of pre- and post-implantation loss, and fetal alterations were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations, and litter size were evaluated using a nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios were analyzed using a binomial distribution test. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and if excluded, were excluded from analysis for documented, scientifically sound reasons. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experimentwise alpha at 0.05. Both were reported at the experiment-wise alpha level.

Reproductive indices:

Calculation of Pre- and Post-Implantation Loss
Pre-implantation loss* = [(No. corpora lutea-implantations) x 100] / No. corpora lutea
Post-implantation loss* = [(No. implantations – viable fetuses) x 100] / No. implantations
* Note: Percent pre- and post-implantation loss was determined for each litter, followed by calculation
of the mean of these litter values

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects on hematology in the 1000 mg/kg/day group consisted of decreases relative to controls in reticulocyte counts (- 38.1 %). Absolute and differential white blood cell counts did not reveal any treatment-related differences at any dose level. There were no treatment-related effects on hematology parameters at 100 or 300 mg/kg/day.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

There were no significant treatment-related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights, or gravid uterine weights at any dose level.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:

other:

Basis for effect level:

other: Not examined

Results: F1 generation

Effect levels (F1)

Dose descriptor:

other:

Basis for effect level:

other: Not examined

Results: F2 generation

Effect levels (F2)

Dose descriptor:

other:

Basis for effect level:

other: Not examined

Applicant's summary and conclusion

Conclusions:

Dams given 1000 mg/kg/day had treatment-related decreases in reticulocyte counts. There were no treatment-related effects on hematology parameters for dams in the 100 or 300 mg/kg/day dose groups. Treatment-related gross pathological observations were limited to the stomach of two of 24 dams given 300 mg/kg/day and 22 of 24 dams given 1000 mg/kg/day and consisted of a thickening of the limiting ridge. There were no treatment-related effects on clinical signs, body weights, body weight gains or feed consumption for any treatment group. There was no indication of embryo/fetal toxicity or teratogenicity at any dose level tested. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day based on the treatment-related decreases in reticulocyte counts. The embryo/fetal no-observed-effect level (NOEL) was 1000 mg/kg/day.

Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity potential of the reaction product of 2-Hydroxyethyl methacrylate (HEMA) and polyphosphoric acid (PPA), hereafter referred to as HEMA phosphate, in Crl:CD(SD) rats following repeated gavage administration. Groups of 24 time-mated female Crl:CD(SD) rats were administered HEMA phosphate by gavage at targeted dose levels of 0, 100, 300, or 1000 mg/kg/day on gestation day (GD) 6-20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 21 blood samples were obtained for a hematological evaluation, and rats were then euthanized, and examined for gross pathological alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral alterations while skeletal examinations were conducted on the remaining fetuses.

Dams given 1000 mg/kg/day had treatment-related decreases in reticulocyte counts. There were no treatment-related effects on hematology parameters for dams in the 100 or 300 mg/kg/day dose groups.

Treatment-related gross pathological observations were limited to the stomach of two of 24 dams given 300 mg/kg/day and 22 of 24 dams given 1000 mg/kg/day and consisted of a thickening of the limiting ridge.

There were no treatment-related effects on clinical signs, body weights, body weight gains or feed consumption for any treatment group. There was no indication of embryo/fetal toxicity or teratogenicity at any dose level tested.

Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 300 mg/kg/day based on the treatment-related decreases in reticulocyte counts. The embryo/fetal no-observed-effect level (NOEL) was 1000 mg/kg/day.