Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A standard Guideline study conducted according to GLP. Well reported.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Material Name: 1,3-Propylenediaminetetraacetic acid
Synonyms: PDTA
Supplier, City, State (lot number): Hampshire Chemical Corporation, Nashua, NH (710P195014-185)
Purity/Characterization (method of analysis and reference) : A purity of 99.1% was determined by averaging triplicate chelometric titrations. Infrared
spectroscopy and nuclear magnetic resonance spectrometry were used to confirm the structure of PDTA. These spectra were consistent with the proposed structure (Malloy, etal., 1999).

Appearance (physical state, color) :Powder (off-white)
Molecular Formula: C11H18N2O8
Molecular Weight: 306.3

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Age at Study Start: Approximately 8 weeks
Strain and Justification
CD rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Physical/Acclimation
Each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes.
Housed 2 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 14 days prior to the start of the study.

Housing
During the study, animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle).
A 12- hour light/dark photocycle was maintained for all animal rooms, with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged at 12-15 times/hour. Cages had wiremesh floors and were suspended above catch pans.
Cages contained a feed crock and a pressure activated nipple-type watering system.
From day 19 of gestation and throughout the lactation phase of the study, females were housed in plastic cages provided with ground corn-cob nesting material.
Room temperature and relative humidity were recorded daily. The relative humidity was maintained within a range of 40-70%. The room temperature was maintained within the range of 19-25°C. These values were within the laboratory recommended range for rats.

Randomization and Identification
Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to yield approximately equal mean body weights and variances per treatment group. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, DE) which were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided Purina Certified Rodent Lab Diet #5002 (Purina Mills, Inc., St. Louis, MO) in meal form. Feed and municipal water were provided ad libitum. Analysis of the feed was performed by Purina Mills, Inc. to confirm that 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 are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company. The results of the feed and water analyses indicated no contaminants at levels that would interfere with the conduct of this study or interpretation of the results.

Animal Welfare
In response to the Final Rules amending the U.S. Animal Welfare Act that were promulgated by the U.S. Department of Agriculture effective October 30, 1989, the Animal Care and Use Activities (ACUA) that were required for the conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC determined that the proposed Activities were in full accordance with these Final Rules.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Dose Preparation

Diets were prepared by serially diluting a concentrated test material-feed mixture (premix) with ground feed. Premixes were prepared periodically throughout the study based on stability data. Diets were prepared weekly for approximately 10 weeks prior to breeding of the P1 adults. The concentration of the test material in the diets was calculated from the most recent body weight and feed consumption data. Initial concentrations of test material in the diet were calculated from pre-study body weights and feed consumption data. To avoid potential overdosing during the breeding period, animals co-housed were provided with the female diets for each dose group. During gestation, females from each dose group were provided with the appropriate dietary concentration of PDTA given during breeding. Dietary concentrations supplied during lactation were adjusted using historical control feed consumption data for lactating females to account for large and rapid increase in food consumption (2-3X increase) typical for rats in late lactation. Until all litters finished the lactation phase, dams awaiting necropsy received a diet containing the same concentration of PDTA that was given during breeding.
Details on mating procedure:
Breeding of the P1 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered day 0 of gestation. The sperm/plug-positive (presumed pregnant) females were then removed from the males’ cages and returned to their individual cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. In cases where a mating partner was not available due to removal from study, the animal was paired with the next available partner.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis
Homogeneity
Representative test diets were evaluated to ensure homogeneous distribution of the test material in the feed at least once during the study.
Stability
The stability of the test material in the vehicle was determined concurrent with the study. The samples included premix, low-dose female and high-dose male diets.
Concentration Verification
Analysis of all test diets to determine concentration of the test material was conducted at least three times. The method used for analyzing the test material in feed included solvent extraction followed by analysis using HPLC with ultraviolet detection and external standards.
Retainer Samples
Reference samples (1/sex/dose/mix and premix) were retained and stored in sealed vials in a manner consistent with the sample retention policy of the laboratory.
Duration of treatment / exposure:
Dosing with PDTA for 10 weeks prior to breeding, and continuing through breeding (2 weeks), gestation (3 weeks) and lactation (3 weeks) to evaluate the potential for reproductive toxicity and effects on neonatal growth and survival.
Frequency of treatment:
Daily in the feed
Details on study schedule:
Breeding occured at 10 weeks after dosing initiation and lasted for 2 weeks.
There was no breeding of the F1 generation.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10 mg/kg bw
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
60 mg/kg bw
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
nominal in diet
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
Justification for dsoe selection:
These dose levels were based on previously discussed data from subchronic studies, in which dose levels of 100 and 300 mg/kg/day in female rats caused increases in urinary zinc excretion, but no changes in serum zinc or other zinc-sensitive parameters. Higher levels of PDTA caused overt signs of zinc deficiency. Since zinc deficiency is a well known cause of reproductive and developmental effects, higher dose levels of PDTA which were likely to result in clinically significant zinc deficiency were not warranted, as they would have confounded study interpretation due to indirect, secondary effects of zinc deficiency. Therefore, 300 mg/kg/day was selected as the top dose level for this one-generation reproduction study. Dose levels of 60 and 10 mg/kg/day were chosen to establish doseresponses for any effects observed and to establish a no-observable-effect-level (NOEL).
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Clinical examinations were conducted on all animals prior to the start of the study and weekly throughout the study period. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses and animal behavior. Twice each day a cageside examination was conducted and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water. Moribund animals that were not expected to survive until the next observation period, were necropsied on that day.

Body Weights
All rats were weighed during the pre-study period and weekly during the 10 week prebreeding treatment. Body weights for males were recorded weekly throughout the study. During the breeding period, female body weights were recorded on the day females were determined to be sperm/plug positive (gestation day 0). Thereafter, gestational body weights were recorded on day 7, 14 and 21 of gestation. Females that delivered litters were weighed on day 1, 4, 7, 14, and 21 of lactation. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases of the study.

Feed Consumption
Feed consumption was determined pre-study and weekly during the 10 week prebreeding treatment period for all animals by weighing feed crocks at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption was measured weekly in males and dietary concentrations were adjusted accordingly. During gestation, feed consumption was measured at weekly intervals for sperm/plug-positive females. Feed consumption was not recorded for females that failed to mate. After parturition, feed consumption was measured on day 1, 4, 7, 11, 14, 17, 19 and 21 of lactation. Feed consumption was not measured in females that failed to deliver a litter.

Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed crock - final weight of feed crock) / (# of days in measurement cycle)
Litter observations:
Litter Data
Females were observed for signs of parturition beginning on or about day 20 of gestation. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of delivery was recorded as the first day the presence of the litter was noted and was designated as lactation day 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (day 0), the number of live and dead pups on day 0, 1, 4, 7, 14, and 21 postpartum, and the sex and the weight of each pup on day 1, 4 (before culling), 7, 14, and 21 of lactation. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. Any pups found dead were examined grossly for external and visceral defects then were
preserved in neutral, phosphate-buffered 10% formalin.

Culling
To reduce the variation in the growth of the pups, F1 litters with more than eight pups were culled on day 4 postpartum. At the time of weighing, each litter of pups was randomly ordered by sex. Culled litters were reduced to a total of 8 pups, 4 males and 4 females, if possible. Cull pups were selected using a computer generated randomization procedure. Litters with 8 or fewer pups were not culled. Preferential culling of runts was not performed. All cull pups were euthanized with Socumb euthanasia solution (Veterinary Laboratories, Inc. Lenexa, KS), examined grossly for external and visceral defects, then were discarded.
Postmortem examinations (parental animals):
Adult Necropsy
A complete necropsy of P1 adults from the control, 10 and 60 mg/kg/day was performed after completion of the lactation phase for females, and approximately the same time for males. A complete necropsy was performed on all surviving 300 mg/kg/day P1 adults prior to completion of the lactation period due to excessive toxicity. All F1 litters except the high-dose litters were euthanized by CO2 inhalation on day 21 postpartum. The highdose litters were euthanized by CO2 inhalation at the time their respective dams were necropsied. Fasted adult rats submitted alive for necropsy were anesthetized by the inhalation of methoxyflurane vapors, their tracheas were exposed and clamped, and the animals were euthanized by decapitation. Due to early termination of the high-dose females (some still had pups), these dams were not fasted prior to necropsy.

The necropsy was conducted by a veterinary pathologist assisted by a team of trained individuals and 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. The eyes were examined in situ by application of a moistened microscope slide to each cornea. 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 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 uteri of all females were examined for the presence of implantation sites. The uteri of females that did not deliver a litter and had no visible implantation sites were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Representative samples of tissues listed below were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes and epididymides were preserved by immersion in Bouin’s fixative. Similar necropsy procedures were followed for animals terminated prior to the scheduled necropsy due to moribund condition; however, the terminal body weights were not recorded and the testes and epididymides were preserved in formalin. Transponders were removed and placed in jars with the tissues.

ADRENALS
KIDNEYS **
PROSTATE **
AORTA
LACRIMAL/HARDERIAN GLANDS
RECTUM
AUDITORY SEBACEOUS GLANDS
LARYNX
SALIVARY GLANDS
BONE (INCLUDING JOINT)
LIVER **
SEMINAL VESICLES **
BONE MARROW
LUNGS
SKELETAL MUSCLE
BRAIN (CEREBRUM, BRAINSTEM, CEREBELLUM)
MAMMARY GLAND **
SKIN AND SUBCUTIS**
CECUM
MEDIASTINAL LYMPH NODE
SPINAL CORD (CERVICAL, THORACIC, LUMBAR)
CERVIX **
MEDIASTINAL TISSUES
SPLEEN
COAGULATING GLANDS **
MESENTERIC LYMPH NODE
STOMACH**
COLON
MESENTERIC TISSUES
TESTES **
DUODENUM
NASAL TISSUES
THYMUS
EPIDIDYMIDES **
ORAL TISSUES
THYROID GLAND
ESOPHAGUS **
OVARIES **
TONGUE
EYES
OVIDUCTS **
TRACHEA
GROSS LESIONS **
PANCREAS
URINARY BLADDER
HEART
PARATHYROID GLANDS
UTERUS **
ILEUM
PERIPHERAL NERVE
VAGINA **
JEJUNUM PITUITARY **
**Reproductive and potential target tissues selected for histopathological examination.

Zinc Determination
During the last week of the pre-breeding phase, urine and blood samples were collected from the first 15 nonfasted rats/sex/group that donated an adequate urine sample in order to determine zinc status. Urine was collected by manual compression of the bladder and was stored at -80 qC. For blood collection, the animals were lightly anesthetized with methoxyflurane and blood samples were collected from the orbital sinus. Serum was prepared and then stored at -80 qC until analysis for zinc content. Zinc concentrations were determined by Inductively Coupled Plasma Atomic Emmision Spectroscopy by Dow Analytical Sciences.

Histopathology
Histologic examination of the adult rats included the reproductive tissues, kidney, liver, esophagus (as an indicator of zinc status), stomach and skin (hind feet) and appropriate gross lesions of all control and high-dose rats as listed above. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. Examination of tissues from the 60 mg PDTA/kg/day group was limited to testes, epididymides, esophagus, stomach, and skin (hind feet), as those tissues demonstrated treatment-related histopathologic effects at the high dose, and relevant gross lesions. Rats found moribund were histologically examined in a similar manner.

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 normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was not 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 was 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 were not regarded as life threatening. A severe grade was 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 have been life threatening.

PDTA ingestion resulted in characteristic histopathologic lesions in selected organs. The grading scheme for these lesions was consistent with the general criteria presented above but specific criteria were developed by the study pathologist as follows:

Stratified squamous epithelium of the esophagus, stomach (non-glandular mucosa), skin (hind feet):
Hyperplasia or Hyperkeratosis:
- very slight – increased thickness, but less than 150% of normal thickness (i.e., 50% increase above the normal thickness) of the non-keratined layer of these organs from control rats;
- slight – estimated to be 150 to 200% of normal thickness;
- moderate – estimated to be 200 to 250% of normal thickness;
- severe – greater than 250% of normal thickness.
Rete pegs were more pronounced as severity grade increased and were particularly evident in the moderate and severe grades.
Parakeratosis:
- very slight – less than three superficial cell layers having retained nuclei;
- slight – three to five layers of nucleated cells;
- moderate – greater than five cell layers.

Testes
Atrophy, seminiferous tubule:
- very slight – less than 10% of the tubules in the testes (combined) were affected;
- slight - 11 to 25% affected;
- moderate – 26 to 50% affected;
- severe – greater than 50% of tubules were atrophic.

Degeneration, seminiferous tubule:
- very slight – up to 25% of non-atrophic tubules were involved;
- slight - 26 – 50% of non-atrophic tubules were involved and the morphologic changes were more prominent;
- moderate - greater than 50% of non-atrophic seminiferous tubules affected.
Degeneration was not used if the degree of atrophy was severe as the number of functional tubules was considered too few to evaluate.

Epididymides
Decreased spermatic elements:
- slight – the spermatic elements were generally less densely stained, acellular space was more evident and retraction zones were larger than controls;
- moderate – paler eosinophilic staining of the spermatidic elements with larger acellular areas and retraction zones such that the area occupied by
the spermatic elements was estimated to be reduced by 26 to 50% of the controls;
- severe – although still present, very few spermatic elements remained in the lumen with greater than 50% reduction in estimated area of spermatic elements.

Aspermia:
No severity grading was recorded because this change represents the most severe end point of the decreased spermatic elements as graded above.
Atypical cells, lumen:
- very slight –atypical cells were present in at least 25% of the tubules but only individual cells or low numbers of cells were present;
- slight – atypical cells were present in greater than 25% of the tubules and multiple atypical cells were often present.

Atypical cells, lumen:
- very slight –atypical cells were present in at least 25% of the tubules but only individual cells or low numbers of cells were present;
- slight – atypical cells were present in greater than 25% of the tubules and multiple atypical cells were often present.
Postmortem examinations (offspring):
Gross exmination only.
Statistics:
Body weights, body weight gains, feed consumption and zinc levels were evaluated by Bartlett's test for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, a Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Gestation length, average time to mating and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Statistical outliers were identified by the sequential method of Grubbs (1969) and were routinely excluded from feed consumptiononly. Outliers for body weight, gestation length, litter size, zinc levels and average time to mating were only excluded from analysis for documented, scientifically sound reasons.
The fertility indices were analyzed by the Fisher’s exact probability test with Bonferroni's correction. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates was analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel (1974) with Bonferroni’s correction. Both the Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experiment-wise alpha level.Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) were greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

In-life Observations
Examinations performed on the rats prior to the study start revealed that all animals were in good health for study purposes. During the study, four male parental rats and one female rat given 300 mg PDTA/kg/day were found to be to be in moribund condition and were terminated prior to the scheduled necropsy. All males had received the test diets for most of the prebreeding period (necropsied on study day 62 or 70) or into the breeding period (day 98 or 100). The four males exhibited extensive body soiling involving the perineal, and perioral and/or perinasal areas, ungroomed and thin appearance, and arched back. Isolated observations, each recorded in separate animals, included decreased activity, decreased feces, and skin flaking and scaling of the hind feet. At necropsy these high-dose males were generally cachexic, most or all had soiling of the fur, decreased adipose tissue, decreased ingesta in the gastrointestinal tract, thickened nonglandular mucosa of the stomach and testes of decreased size or flaccid. One male had gastric erosions or ulcers with hemolyzed blood in the gastrointestinal tract. Subsequent histological examination of these animals revealedstomach and esophageal lesions that likely contributed to the cachexic condition with stomach hyperkeratosis graded as severe in all rats, while two had atrophy of the nonglandular mucosa and erosions and ulcers of the glandular mucosa. In addition, three rats had moderate esophageal parakeratosis. All four rats had severe atrophy of the testes. A single female from the 300 mg/kg/day dose group was found in a moribund condition and was necropsied on test day 29. Earlier in the study, this animal was noted to have maloccluded incisors. One week after this observation was recorded, this animal exhibited red periocular soiling, pale skin and mucous membranes, labored respiration and an apparent mechanical injury to the muzzle. At necropsy, this animal was found to have a broken maxilla and gas in the gastrointestinal tract. In conjunction with the maxilla fracture, severe fibrinopurulent inflammation of the nasal tissues was observed histologically. The necropsy findings for this female were distinctly different from those noted for the males and were consistent with accidental trauma with compromise of its airway.

All other parental rats survived until the scheduled necropsy. Treatment-related clinical observations recorded in the surviving high dose animals included thin appearance, ungroomed appearance, dermatitis, alopecia, red perioral soiling, red perinasal soiling, and abdominal urine soiling. These signs were markedly more prevalent in male than in female animals. Males were noted to be continuously grooming their forepaws, an observation that may be related to extensive perioral/perinasal soiling. Arched back, flaking and scaling of skin on the hind paws, perineal soiling (urine), and extensive body soiling (urine or urine and feces) were noted in male animals only. Near termination, increased reactivity in response to handling (most likely related to their poor condition) was exhibited by all male and three female rats in the high dose group. These clinical observations in the high dose group, particularly lesions involving the integument (e.g. alopecia and flaking/scaling of the paws) are consistent with clinical zinc deficiency (Hamdi et al., 1997). No treatment-related effects on clinical observations were seen in males or female rats given 60 or 10 mg/kg/day

Feed Consumption
Males in the 300 mg/kg/day group consumed significantly less feed than controls, starting from the second week of exposure and continuing through the end of the study. Feed consumption of the 300 mg/kg/day females was generally comparable to controls during the pre-breeding period, as well as the first two weeks of gestation, but was significantly decreased during the last week of gestation and during the lactation phase of the study. Feed consumption of the males and females of the 60 and 10 mg/kg/day groups was comparable to controls throughout the study with the exception of a statistically identified decrease for the 10 mg/kg/day females on lactation days 7-11 and 19-21. These decreases were not considered to be treatment related as there were no significant changes in lactation feed consumption in dams given 60 mg/kg/day.

Body Weights
Body weights of the 300 mg/kg/day group males were significantly lower than controls, starting from test day 13 of the pre-breeding period, and continuing for the remainder of the study. In fact, mean body weights actually decreased from test day 48-69, although this decrease was mainly due to the influence of two males that were subsequently sacrificed in moribund condition. Mean body weight losses of the 300 mg/kg/day males also were seen after test day 83, but in this case, all of the males in this group exhibited weight loss. By the time of necropsy on test day 106, these males weighed approximately 33% less than their contemporary controls. In the 300 mg/kg/day group females, body weights during the pre-breeding exposure period were also decreased, albeit to a lesser extent than the males. Body weights of the top dose females continued to be decreased throughout gestation (statistically identified only on gestation day 21) and throughout lactation. Corresponding decreases in body weight gains of the females in this group were statistically identified on gestation days 14-21. During lactation, a statistically identified increase in body weight gain was found on lactation days 7-14, but was not considered of toxicological significance. Body weight and body weight gain of the males and females of the 60 and 10 mg/kg/day groups were comparable to controls throughout the study.

Serum and Urine Zinc
Urine zinc levels were significantly elevated in the male rats of the 60 and 300 mg/kg/day group, and in female rats at all dose levels of PDTA. This increased excretion of zinc was expected of a chelating agent, such as PDTA. In and of itself, increased urinary excretion of zinc is not considered a toxicologic effect, because increases in urine zinc can occur without compromise of zinc homeostasis. In fact, serum zinc is generally accepted as a more relevant measure of overall zinc homeostasis (King, 1990). Consistent with this, serum zinc values were normal in both males and females of the low and middle-dose groups. However, serum zinc values in the high dose males and females were significantly lower than those of the controls, and indicated a marked zinc deficiency in this dose group. This serum zinc profile is consistent with the histopathological and reproductive effects seen only in the high dose group animals (discussed below).

Gross Pathology
At the scheduled necropsy, a variety of observations were made for male rats given 300 mg PDTA/kg/day that were considered related to PDTA ingestion. The majority of males had decreased adipose tissue, facial soiling and scales on the rear feet. Other effects attributed to PDTA ingestion and noted in some males, from 5 to 9 of the 26 males in this group, included thickened non-glandular mucosa of the stomach, erosions and ulcers of the stomach, and flaccid or decreased size of the testes. The only apparent treatment related effect in females given 300 mg/kg/day was increased numbers of nonpregnant rats.

Several other observations were noted in a low numbers of rats in a random manner with both controls or rats given any of the PDTA dose levels affected. These changes were considered spontaneous changes and were considered typical for rats of this strain, age and husbandry conditions.

Histopathology
Treatment-related histopathologic effects were identified in several organs from rats given 300 mg PDTA/kg/day and terminated at the scheduled necropsy. The stratified squamous epithelium of the stomach (non-glandular portion), esophagus, and the rear feet was affected in both males and females given this dose level. The effects were termed hyperplasia, hyperkeratosis and parakeratosis. These changes were qualitatively similar for all organs but the relative response was quantitatively different among the organs.

Hyperplasia of the stratified squamous epithelium was noted in the esophagus, stomach and hind feet of almost all males and females given 300 mg/kg/day. Hyperplasia referred to increased cell number and thickness of the viable epithelial cell layers, usually with increased basophilia of the basilar epithelium, and accentuation of the rete pegs. Hyperplasia was generally of slight grade in the esophagus and was noted for almost all high dose male and female rats from the scheduled termination in approximately equal severity. Hyperplasia of the epidermis of the rear feet (plantar surface or foot pad) was present in all males and most females from this group but was of greater severity in males. Males had a greater effect in the non-glandular mucosa of the stomach with 23 of 26 affected, including 10 of moderate degree and 2 of severe grade. For females given 300 mg PDTA/kg/day, only 6 of 29 had hyperplasia of the non-glandular mucosa of the stomach, of which 5 were graded very slight.

Hyperkeratosis, increased thickness of the superficial keratinized layer comprised of flattened epithelial cells lacking nuclei and generally dense eosinophilic staining, was noted primarily in the stomach and hind feet but only rarely in the esophagus. Males were much more affected with hyperkeratosis, particularly in the stomach. In the stomach, 25 of 26 males had hyperkeratosis while only 4 of 29 females had this change, of which 3 were very slight. Hyperkeratosis of the rear feet was found for 20 males and 10 females given 300 mg PDTA/kg/day although the severity was similar with the greatest frequency being slight for either sex.

Parakeratosis, a somewhat unique lesion characterized by retention of nuclei in the superficial keratinized layer, was a prominent lesion of the esophagus of both males and females and was also common in the rear feet of males but noted only once in the stomach. Parakeratosis was found in the esophagus of 25 of 26 males and 18 of 29 females given 300 mg PDTA/kg/day. Additionally, the males had greater severity. Parakeratosis was found in the rear feet of 25 males but only 7 females.

Testes and, secondarily the epididymides, were affected in males given 300 mg PDTA/kg/day. Spermatogenesis was altered with the effect termed degeneration for those seminiferous tubules in which spermatogenesis was altered but was still present or atrophy for tubules lacking spermatogenesis. Degeneration was characterized by a constellation of changes in the germinal epithelium comprised of the following morphologic components (in approximate order of frequency): 1. large vacuoles, usually near the basilar portion of the germinal epithelium; 2. abnormal spermatogenesis with spermatocytes in the lumen, less organization or irregular thickness of the germinal epithelium within an individual tubule, and sometimes multinucleated spermatids; and 3. dilated tubule lumen. Atrophic seminiferous tubules were small and composed solely ofpale eosinophilic, columnar cells with ill-defined cell boundaries, consistent with Sertoli cells. Both changes were graded based upon the estimated percentage of affected tubules (see methods). Degeneration was not used if the degree of atrophy was severe as the number of functional tubules was considered too few to evaluate. All males given 300 mg PDTA/kg/day that were terminated at the scheduled necropsy had effects upon the testes, with 8 considered to be severe atrophy. The remaining 18 rats had varying grades of degeneration and atrophy of the seminiferous tubules. Three control males and three males given 60 mg PDTA/kg/day had very slight histopathologic effects in the testes, which were considered to be spontaneous changes. Consistent with the effects on spermatagenesis noted for the testes, decreased or absent (aspermia) spermatids along with increased abnormal cells were noted in the epididymides of most males given 300 mg PDTA/kg/day. There were no effects noted for those given 60 mg PDTA/kg/day.

The only treatment-related effect found in female reproductive organs was decreased numbers of females given 300 mg PDTA/kg/day that had foci of pigmented macrophages in the uterus. These represent the former sites of placental implantation and this finding is consistent with the decreased number of pregnant rats in this dose level. There were low numbers of other histologic changes noted in these organs as well as the other organs examined that were considered incidental lesions. These generally occurred in low numbers of rats, were seen in a random pattern with some controls affected, and were generally of minimal severity.

Reproductive Indices, Pup Survival, and Sex Ratio
There were no treatment-related effects at any dose level on parameters related to mating performance, i.e., the number of days to mating, male mating index, or female mating index. While the mean number of days to mating in the middle and high dose groups (2.8 days) was slightly higher than that of controls (2.1 days), the control value was unusually low and outside the historical control range. The middle and high-dose values were well within the historical control range (Table 23 and Appendix Table 18). Similarly, gestation length was similar across all groups. Appendix Tables 15-17 contain individual data for these reproductive parameters.

Although there were no apparent effects on mating performance, there were marked effects on fertility, gestation survival and pup survival in the 300 mg/kg/day rats. Male and female fertility indices, male and female conception indices, gestation survival index, and pup survival indices on post-natal day 1 and 4 all were significantly decreased in the 300 mg/kg/day dose group, with only 11 litters being produced in this group. Although the one-generation study is not designed to determine the affected sex responsible for reproductive effects, the marked testicular toxicity as an apparent consequence of zinc deficiency would appear to be the major cause of reduced fertility at the high dose. Although pup survival did not appear to be affected after post-natal day 4, there were only 15 pups remaining in this high dose group at these time points. No pup survival data are provided for the day 21 time point, due to the change in termination schedule for this dose group. In contrast to the effects on fertility and gestation/lactation survival seen at the top dose level, there were no effects on any parameter of reproductive performance or survival in the middle and low dose groups. There were no effects on pup sex ratio at any dose level tested.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: Parental (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

Litter Observations
Treatment-related observations noted in pups from the 300 mg/kg/day treatment group were decreased pup survival (see text above) with a higher occurrence of cannibalization and an increased incidence of pups that were cold to touch, thin and dehydrated, and lacked milk in their stomachs. Some high dose pups were found with their placenta still attached. These observations suggest alterations in maternal caregiving behavior. Attached placentae and the absence of milk in the pups’ stomachs also were noted in two pups in one litter from the mid-dose group. Other observations recorded in the offspring appeared at low frequency and were not treatment related.

Litter Size and Pup Body Weights
Consistent with the aforementioned effects on fertility and gestation/lactation survival, litter sizes in the 300 mg/kg/day dose group were significantly decreased at all time points. There was also a statistically significant increase in the number of pups born dead in this group. Body weight data for the surviving high dose group pups was somewhat difficult to evaluate due to the small number of litters and reduced litter sizes in this group. Nonetheless, pup body weight values in the high dose group appeared to be similar to those of the control group, with the exception of a statistically significant decrease in female pup body weights only on postnatal day 11. The significance of this single decrease is equivocal. Again, in contrast to effects seen in the 300 mg/kg/day dose group, litter sizes and pup body weights in the 60 and 10 mg/kg/day groups were unaffected by treatment.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Relationship of Histopathological and Reproductive Effects to Zinc Deficiency

In the current study, PDTA at dose levels of 10 and 60 mg/kg/day in the diets of CD rats resulted in no adverse effects, but clearly resulted in a clinical zinc deficiency accompanied by histopathological and reproductive effects at a dose level of 300 mg/kg/day. The latter results were unexpected, based on a prior 14-week toxicity study in which doses of up to 300 mg/kg/day of PDTA via gavage produced no effects on body weights, feed consumption, hematology, clinical chemistries, gross pathology, histopathology or organ weights. Although increased urinary zinc excretion was noted, serum zinc levels remained normal (Gordon and Krasavage, 1985). Both the histopathological and reproductive effects seen in the current study were highly consistent with the well characterized effects of zinc deficiency, and thus, were considered secondary to compromised zinc homeostasis caused by continuous high dose exposure to the chelating agent, PDTA. In particular, hyperkeratosis, parakeratosis and hyperplasia of the stratified squamous epithelium of the esophagus, stomach and foot pads seen in this study are classic signs of zinc deficiency (Diamond et al., 1971). Similarly, testicular effects resulting from zinc deficiency in rats have been well characterized and were very consistent with the effects seen in this study (Diamond et al., 1971; McClain et al., 1984; Merker and Gunther, 1997; Hamdi et al., 1997). Zinc deficiency is also known to interfere with normal parturition and maternal care during the early post-natal period (Apgar, 1977), which again is consistent with the decreased pup survival and increased stillborn pups seen in this study.

Applicant's summary and conclusion

Conclusions:
PDTA, a chelating agent, was associated with alterations in zinc homeostasis at the high dose level of 300 mg/kg/day, as evidenced by decreased serum zinc levels, increased urinary zinc excetion, clinical/gross observations of hind foot scaling, and histopathological findings such as hyperplasia, hyperkeratosis and/or parakeratosis of the esophagus, stomach and skin, and testicular changes (degeneration and/or atrophy of the seminiferous tubules, decreased or absent spermatids). All of these effects are known to be associated with zinc deficiency in rats. Four male rats in this group were sacrificed in moribund condition prior to the scheduled termination and had signs of marked zinc deficiency at necropsy. Decreases in feed consumption and body weights were noted in both males and females, with males being more severely affected (top dose males weighed 33% less than controls at termination). Accompanying the compromised zinc status at the top dose level were marked decreases in fertility and pup survival. No adverse effects of PDTA on systemic or reproductive toxicity were found in the 60 or 10 mg/kg/day groups. Although statistically identified increases in urinary zinc excretion were noted at 60 mg/kg/day (both sexes) and 10 mg/kg/day (females only), this was not considered an adverse effect, as serum zinc levels and other zinc-sensitive parameters were unaltered. The no-observable-adverse-effect-level (NOAEL) for systemic and reproductive effects in this study was 60 mg/kg/day.
Executive summary:

Groups of 30 male and 30 female CD rats were fed diets supplying 0, 10, 60 or 300 mg/kg/day of propylenediaminetetraacetic acid (PDTA) continuously for 10 weeks prior to breeding and during breeding, gestation and lactation to evaluate the potential for reproductive toxicity and effects on neonatal growth and survival. In-life observations, body weights, feed consumption and litter data were evaluated. In addition, a gross necropsy of the P1 adults was conducted with extensive histopathologic examination of tissues.

PDTA, a chelating agent, was associated with alterations in zinc homeostasis at the high dose level of 300 mg/kg/day, as evidenced by decreased serum zinc levels, increased urinary zinc excetion, clinical/gross observations of hind foot scaling, and histopathological findings such as hyperplasia, keratosis and/or parakeratosis of the esophagus, stomach and skin, and testicular changes (degeneration and/or atrophy of the seminiferous tubules, decreased or absent spermatids). All of these effects are known to be associated with zinc deficiency in rats. Four male rats in this group were sacrificed in moribund condition and had signs of marked zinc deficiency at necropsy. Decreases in feed consumption and body weights were noted in both males and females, with males being more severely affected (top dose males weighed 33% less than controls at termination). Accompanying the compromised zinc status at the top dose level were marked decreases in fertility and pup survival. No adverse effects of PDTA on systemic or reproductive toxicity were found in the 60 or 10 mg/kg/day groups. Although statistically identified increases in urinary zinc excretion were noted at 60 mg/kg/day (both sexes) and 10 mg/kg/day (females only), this was not considered an adverse effect, as serum zinc levels and other zinc-sensitive parameters were unaltered. The no-observable-adverseeffect- level (NOAEL) for systemic and reproductive effects in this study was 60 mg/kg/day.