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Administrative data

Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information

Developmental toxicity of HEMA phosphate was examined in rats by oral gavage at dose levels of 100, 300, and 1000 mg/kg/day. The maternal NOAEL was 300 mg/kg/day based on treat-related decreases in reticulocyte counts observed at 1000 mg/kg/day. There was no indication of embryo/fetal toxicity or teratogenicity at any dose level tested. The NOEL for embryo/fetal toxicity was 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 14, 2016 - March 9, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: JMAFF Guideline 2-1-18, Teratogenicity Study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Test material determined to be stable under storage conditions for 22 days by HPLC.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals 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.
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 MKBV7805V, 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation analyses of all dose levels were determined pre-exposure. The homogeneity of the low-dose and the high-dose test solutions were 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). Analysis of all dosing solutions from the first mix revealed acceptable mean concentrations of HEMA phosphate ranging from 95.5% to 96.9% of targeted concentrations. Analyses of the low- and high-dose dose solutions indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 2.0%.

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. Therefore, additional stability analyses in PG were not conducted.

The stability of the neat test material (NTM) was assessed by generating and comparing a minimum of three replicate samples. The replicate samples were generated from discrete preparations (NTM used for each preparation) of stock solutions. The intended stability would cover the duration from the initial dispenseand use of the NTM to the final dispense and use of the NTM for this study (23 days). Inadvertently, the actual NTM stability duration covered was 22 days. The HEMA phosphate day 0 NTM stability data (average purity) was 91.0% ± 0.286% (n=3) and the day 22 NTM stability data (average purity) was 90.5% ± 0.164% (n=3) indicating no change in HEMA phosphate purity over a 22 day period.

Solubility was confirmed in PG at concentrations up to 500 mg/mL (Andrus et al., 2016).
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.
Duration of treatment / exposure:
gestation day 6 through 20
Frequency of treatment:
daily
Duration of test:
necropsy was performed on gestation day 21
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
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.
Maternal 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.
Ovaries and uterine content:
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.
Fetal examinations:
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.
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.
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
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.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related gross observations were limited to the stomach and consisted of a thickening of the limiting ridge (a fold of the stomach mucosa that demarcates the border between the forestomach and the glandular stomach) in two of 24 dams given 300 and 22 of 24 dams given 1000 mg/kg/day. All other gross findings were interpreted to be spontaneous changes unassociated with exposure to the test material.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
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.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
haematology
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related skeletal alterations in any dose group. Incidental findings bearing no relationship to treatment included the malformations of: sternoschisis in one fetus (dam 52) from the control group and one fetus (dam 127) from the 1000 mg/kg/day dose group, missing thoracic vertebrae, centra and ribs in one fetus (dam 119) from the 1000 mg/kg/day dose group, and a single fetus (dam 90) from the 300 mg/kg/day dose group with an extra thoracic vertebra, centra and ribs (Text Table 5). Additional incidental findings bearing no relationship to treatment included the variations: of delayed ossification (DO) parietal, DO interparietal, DO occipital, DO thoracic centra, DO sternebrae, irregular pattern of ossification sternebrae, calloused ribs, and class I and II wavy ribs. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, they were considered spurious and unrelated to treatment.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included the variations: accessory blood vessel, supernumerary hepatic lobule, and hemorrhage on the adrenal gland. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, they were considered spurious and unrelated to treatment.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed
Developmental effects observed:
no

Table 1: Treatment related hematologic differences

Dose (mg/kg/day)

0

100

300

1000

Reticulocytes count (E9/L)

60.4

50.0

63.0

37.4*

*Statistically different from control mean by Dunnett’s test, alpha = 0/05

Table 2: Incidence of external malformations

Dose (mg/kg/day)

0

100

300

1000

Macroglossia

Fetuses

0/292

1/312

0/276

0/322

Litters

0/22

1/22

0/21

0/24

 

Table 3: Incidences of skeletal malformations

Dose (mg/kg/day)

0

100

300

1000

Sternoschisis

Fetuses

1/147

0/158

0/138

1/160

Litters

1/22

0/22

0/21

1/24

Missing Thoracic Centra

Fetuses

0/147

0/158

0/138

1/160b

Litters

0/22

0/22

0/21

1/24

Missing Thoracic Vertebra

Fetuses

0/147

0/158

0/138

1/160b

Litters

0/22

0/22

0/21

1/24

Missing Ribs

Fetuses

0/147

0/158

0/138

1/160b

Litters

0/22

0/22

0/21

1/24

Extra Ribs

Fetuses

0/147

0/158

1/138a

0/160

Litters

0/22

0/22

1/21

0/24

Extra Thoracic Vertebra

Fetuses

0/147

0/158

1/138a

0/160

Litters

0/22

0/22

1/21

0/24

Extra Thoracic Centra

Fetuses

0/147

0/158

1/138a

0/160

Litters

0/22

0/22

1/21

0/24

a-bMalformations denoted with the same superscript were noted in a single fetus

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
An OECD 414 GLP study is available in rats, the preferred rodent species according to the guideline recommendations. Testing was performed up to the limit dose with no developmental effects observed. This study is adequate to assess the developmental toxicity of HEMA phosphate.

Justification for classification or non-classification

There is no justification for classification based on data from available studies.