Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test article was identified in this study as follows:
[14C]-AMP (molecular weight = 91.1 g/mol; specific activity = 25 mCi/mmol)


[14C]-AMP was provided by The Dow Chemical Company prepared in three formulations, identified in this study as follows:

1. AMP (Batch No. TA074801Z1) – Contains 94.85 mg AMP/g and 0.27 mg 14C-AMP/g, for a total calculated value of 95.12 mg AMP/g (Paste)

2. 40% aqueous solution (w/v, pH 9.5) prepared from AMP - Contains 0.26 mg 14C-AMP/g (Liquid)

3. Control Lotion B with pure AMP (Lot BP200500759.06) – Contains 40 mg AMP/g and 0.26 mg 14C-AMP/g, for a total calculated value of 40.26 mg AMP/g (equivalent to the level of AMP in cosmetic lotions)


Pure AMP, 40% aqueous, and Neat Control Lotion B formulations were evaluated for radiochemical concentration, which was determined to be, respectively, 79.3, 86.2, or 87.6% in a first shipment of formulations, and 80.3, 88.9, or 92.4% in a second shipment of formulations.
Specific details on test material used for the study:
2-methyl-2-aminopropanol hydrochloride salt ( Synonyms: AMP-HCl)
Supplier, City, State (lot, reference number)
Angus Chemical Company, a subsidiary of the Dow Chemical Company, Buffalo Grove,
Illinois (lot# CEC-200303541-12; received 2-5-2004).
Purity/Characterization (method of analysis and reference)
The purity of AMP HCl (lot CEC-200303541-12), corrected for water content (0.16%) was
99.7% as determined by gas chromatography (GC) after the sample was derivatized to
form volatile trimethylsilyl-species. No impurities (other than water) were present at
0.1% or higher. Infrared spectroscopy (IR) and GC/mass spectrometry (GC/MS) were
used to confirm the proposed structure of AMP (Barr et al., 2004).
Doses were based on the AMP HCl salt and were not adjusted for purity.
Characteristics
Appearance (physical state, color)
White crystalline
Molecular Formula
C4H11NO.HCL
Molecular Weight
125.6

AMP HCl is approximately 70% AMP; therefore a dose of 100 mg/kg bw/day AMP HCl corresponds to approximately 70 mg/kg bw/day AMP

Test animals

Species:
rat
Strain:
other: CD
Sex:
male/female
Details on test animals and environmental conditions:
CD (Crl:CD(SD)IGSBR) rats were obtained from a commercial supplier and were approximately eight weeks of age at the time of study initiation. Each animal was evaluated by a laboratory veterinarian or a trained animal/toxicology technician, under the direct supervision of a lab veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately two weeks prior to the start of the study. They were offered a commercial diet and water ad libitum. During the study, animals were housed one per cage (prebreeding) or two per cage (one male and one female during breeding) in stainless steel cage in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Dams were housed one per cage (with their litter) in plastic cages provided with corn cob nesting material from approximately day 19 of gestation and throughout the lactation phase of the study. Animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
Groups of 12 male and 12 female CD rats were fed diets supplying 0 (control), 100, 300, or 1000 mg/kg/day of AMP HCl. Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for 37 days. The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days).
Details on mating procedure:
Breeding of the adults commenced after approximately two 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 the 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 or plug-positive (presumed pregnant) females then were separated from the male and returned to their home cages. If mating did not occur after two weeks, the animals were separated without further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples from the test diets were evaluated concurrently with the concentration verification analyses (see below) to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed at least once during the study. Preliminary stability analyses of the test material in rodent diets at concentrations of 0.0005% and 0.005% and 5.0% were initiated prior to the start of the range-finding study. Analysis of all test diets from the first mix of the main study were initiated prior to the start of dosing using gas chromatography-mass spectrometry (GC-MS) incorporating an internal standard to determine target concentrations.
Duration of treatment / exposure:
Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for 37 days. The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days).
Frequency of treatment:
Continuous
Doses / concentrations
Remarks:
Doses / Concentrations of AMP HCl :0, 100, 300, 1000 mg/kg/day (0, 70, 210, 700 mg/kg/day AMP) Basis:nominal in diet
AMP HCl is approximately 70% AMP; therefore a dose of 100 mg/kg bw/day AMP HCl corresponds to approximately 70 mg/kg bw/day AMP
No. of animals per sex per dose:
12/sex/dose
AMP HCl is approximately 70% AMP; therefore a dose of 100 mg/kg bw/day AMP HCl corresponds to approximately 70 mg/kg bw/day AMP
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 12 male and 12 female CD rats were fed diets supplying 0 (control), 100, 300, or 1000 mg/kg/day of AMP HCl (0, 70, 210, 700 mg/kg/day AMP). Males were exposed for at least two weeks prior to breeding and continuing throughout breeding for 37 days. The females were exposed for two weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days). Effects on gonadal function, mating behavior, conception, development of the conceptus, parturition, litter size, pup survival, sex, pup body weight and the presence of gross external morphological alterations were assessed. In addition, a gross necropsy and histopathology of the adults was conducted with an emphasis on organs of the reproductive system. Males were dosed via the diet for at least 14 days prior to mating, continuing throughout mating, for 37 days. Females were dosed by dietary exposure for 14 days prior to breeding, and continuing through breeding (up to two weeks), gestation (three weeks), and lactation (four days).
Positive control:
no

Examinations

Parental animals: Observations and examinations:
Daily ObservationsA cage-side examination was conducted twice daily, designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals for all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 20. Females that delivered litters were subsequently evaluated on LD 0, 1 and 4, and on additional days if warranted by observations made during daily cage-side examinations. Females that failed to mate or deliver a litter were examined weekly. 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/Body Weight GainsBody weights for males were recorded on test days -1, 1, 4, 7, and weekly thereafter. Females were weighed on test days 1, 4, 7, and 14 during the pre-breeding period. During gestation, females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.Feed ConsumptionFeed consumption for all animals was measured on test days 1, 4, 7 and 14 during the pre-breeding period by weighing feed containers 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 for males. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter.
Litter observations:
Litter DataFemales were observed for signs of parturition beginning on or about GD 20. 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 LD 0. 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 LD 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they are observed during the lactation period. Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, to the extent possible, for external and visceral defects and discarded.
Postmortem examinations (parental animals):
Adult NecropsyA complete necropsy of all the adults was performed. All males were necropsied on test day 38, while females that delivered litters were necropsied on LD 4. Females that did not deliver a litter were necropsied at least 24 days after the last day of the mating period. In all cases, dosing continued until the day prior to sacrifice at which time the animals were fasted overnight. Fasted adult rats submitted alive for necropsy were anesthetized by the inhalation of carbon dioxide, weighed, and their tracheas exposed and clamped. The animals were then euthanized by decapitation.A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues, and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. 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 stained with a 10% solution of sodium sulfide stain for approximately two minutes and were examined for the presence and number of implantation sites (Kopf et al., 1964). After evaluation, the uteri were gently rinsed with saline and preserved in neutral phosphate 10% formalin. Weights of the epididymides, kidneys, liver, and testes were recorded, and organ:body weight ratios calculated. HistopathologyTissues with relevant gross lesions was conducted on all adult rats from the control and high-dose groups. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. Examination of tissues from the remaining groups was limited to the liver (males and females), cervix, ovaries, oviducts, uterus, vagina, and relevant gross lesions. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. The presence and integrity of the 14 stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). 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 would not be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade 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 would not be 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 be life threatening.The above grading criteria were not sufficiently flexible to accurately characterize the liver microscopic vacuolization consistent with fatty change found in this study. Therefore, the following criteria were applied only to the observation of "liver vacuolization, consistent with fatty change, hepatocyte, multifocal": grade 1 - infrequently observed, grade 2 - occasionally observed, and grade 3 - readily observed.
Postmortem examinations (offspring):
Offspring NecropsyAll pups surviving to LD 4 were euthanized by oral administration of sodium pentobarital solution, examined for gross external alterations, and then discarded. Any pups found dead were examined to the extent possible.
Statistics:
see below
Reproductive indices:
Calculation of Reproductive Indices Reproductive indices were calculated for female and male mating indices, male conception index, female and male fertility indices, gestation index, gestation survival index, post-implantation loss, and days 1 and 4 pup survival indices.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dams at 300 mg/kg/day exhibited non-statistically weight decrease
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and relative liver and kidney weights of males doses at 1000 mg/kg/day were statistically identified and were outside of the historical control range , suggesting a treatment-related effect. Similar effects were not seen in females at any dose
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatcellular changes in 12/12 males was observed in the 1000 mg/kg/day, characterized by slight diffuse cyto;plasmic microvacuolization of the periportal heptaocytes. Similar effects were seen in 3/12 control female rats and nearly all of the treated female rats. In the female rats of all treated groups the incidence and severity of hepatic fatty change was increased compared to respective controls
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increases in post-implantation loss (embryo resorption)

Details on results (P0)

All animals survived until scheduled termination. No treatment-related effects on behavior or demeanor were observed at any dose level during the treatment-period.There were no treatment related differences in the amount of feed consumed by any of the treated males or females when compared to their respective controls throughout the study. Gestation feed consumption of dams given 1000 mg/kg/day could not be compared due to a lack of viable litters at this dose level.No significant differences in body weights were observed for males at any dose level tested. Pre-mating body weights of treated females were not different from controls. Dams given 300 mg/kg/day exhibited a non-statistically significant decrease (5.5%) in gestation day 20 body weight, and a statistically significant decrease in body weight gain between GD 14 and 20. This also led to a slight decrease (not statistically identified) in body weight gain for the GD 0-20 interval. The mean GD 14-20 body weight gain in the 300 mg/kg/day group was 40% less than the control body weight gain during this period, which correlated closely with an approximate 50% reduction in mean litter sizes observed in this group. Gestation body weights and body weight gains of dams given 100 mg/kg/day were not different from controls. Gestation body weights and body weight gains of females given 1000 mg/kg/day could not be compared due to a lack of viable litters at this dose level. There were no treatment-related differences in body weight or body weight gains during the lactation phase of the study.There were no treatment-related effects at any dose level on mating, conception, fertility, time to mating, gestation length or sex ratio. However, a marked increase in percent post-implantation loss (embryo resorption) occurred in the 300 and 1000 mg/kg/day group females, with both the incidence, severity and likely timing of this effect being dose-related. In the 1000 mg/kg/day group, all twelve pregnant females showed evidence of complete litter resorption. In the 300 mg/kg/day group, four of twelve females exhibited complete litter loss, with the remaining eight females showing partial litter losses to varying degrees. Judging from the gross and microscopic appearance of the resorbed implantation sites, as well as the pattern of individual animal body weight gains during gestation, it is estimated that the embryonic deaths occurred sometime toward the end of the second week of gestation (implantation is completed on GD 6; full term is 21-22 days), with some of the high dose animals perhaps occurring slightly earlier. In the 100 mg/kg/day group, all reproductive parameters including percent postimplantation loss were comparable to control values. One of 12 females in the 100 mg/kg/day group was initially considered non-pregnant based on the absence of implantation sites following sodium sulfide staining at necropsy. However, histological examination of the uterus revealed a very small amount of hemosiderin pigment in only one implantation site, suggesting that an embryo had implanted and then was resorbed almost immediately. This single case of very early resorption does not fit with the timing of resorption seen in the 300 and 1000 mg/kg/day group, which occurred somewhat later. As pregnancies are not normally diagnosed histologically, there are no historical control data to directly address the significance of this observation. However, the likelihood that this was a spurious finding is supported by the fact that isolated occurrences of females that mate, but do no show evidence of embryo implantation following sodium sulfide staining are commonly encountered in reproductive toxicity studies. Considering this, and the fact that all reproductive parameters in this group were unaffected by treatment, this single incident of litter resorption in the low dose group was considered spurious and unrelated to treatment There were no treatment-related effects on litter size in dams given 100 mg/kg/day. In the 300 mg/kg/day group, there were statistically identified decreases in mean number of pups born live, and mean number of pups on day 1 and 4 postpartum. These decreases in litter size were a function of the increase in post-implantation loss, as noted previously. No litters were produced by the 1000 mg/kg/day group females. In the 1000 mg/kg/day males, mean body weight was increased approximately 5% when compared to the control group. Although not statistically identified, it was associated with statistically identified increases in absolute and relative liver and kidney weight. Similar body or organ weight effects were not observed in any of the female groups. However, it should be noted that terminal body weight and organ weight data from the 1000 mg/kg/day group were excluded from analysis because they did not produce any viable litters and, therefore, could not legitimately be compared to the controls, which were in a post-partum state. Also excluded from analysis were four females in the 300 mg/kg/day group and one in the 100 mg/kg/day group which did not produce viable litters. The increased absolute and relative liver and kidney weights of the 1000 mg/kg/day males were statistically identified and were outside of the historical control range for several studies, suggesting a treatment related effect. There were no treatment-related gross pathologic observations. All animals survived to the scheduled termination of the in-life phase of the study. Males given 1000 mg/kg/day and females given 100 mg/kg/day had treatment-related liver effects. Hepatocellular changes in 12/12 males of the 1000 mg/kg/day group were characterized by very slight, diffuse, cytoplasmic microvacuolization of periportal hepatocytes. In general, the very slight effect was present in most of the periportal hepatocytes; however, in the three liver sections examined from different lobes (left lateral, middle, and right lateral), one was generally more noticeably affected. In general, the left and right lateral lobes were more affected than the middle lobe. The gradation of the effect was based upon the most severely affected lobe. The livers of a few male rats in the control, low and middle dose group were also affected. It is likely the microscopic hepatocellular change in the 1000 mg/kg/day males was related to the significantly increased absolute and relative liver weight observed in this group. There were 3/12 female control rats, and nearly all treated female rats with a similar microscopic hepatic effect. In addition to the aforementioned microscopic liver change in the various groups, another observation characterized by individual hepatocytes with vacuolated cytoplasm, suggestive of fatty change, was noted. The fatty change generally involved either the left or right lateral lobe most extensively. In no case was the middle lobe the most severely involved. In rats with a grade 2 or 3 involvement of hepatocytes with fatty change, the effect extended from the periportal region to the midzone of the lobule. Even in the most severely affected livers of either sex, the hepatocytes surrounding the central vein were microscopically normal. The fatty change in livers of 1000 mg/kg/day males was observed at a higher frequency and severity when compared to lower dose groups and their respective control group. In the female rats of all treated groups, the incidence and severity of hepatic fatty change was increased compared to their respective controls. In the female rats of all treated groups, the incidence and severity of hepatocellular fatty change was increased compared to their respective controls. The number of female rats in the 100 and 300 mg/kg/day groups was more frequently affected with a grade 2 and 3 hepatocellular fatty change in the 1000 mg/kg/day group. This may be due to the fact no litters were being nursed in the highest dose group. A clear dose response for the hepatic effect was not present in female 100 and 300 mg/kg/day groups. The preferential involvement of the left and right lateral lobes verses the middle lobe, in hepatocellular fatty change, may be due to normal variation in portal blood flow and/or normal variation in metabolic enzymes involved in lipid and triglyceride metabolism. The microvacuolization and the fatty change in hepatocytes of even the most severely affected rats were not associated with evidence of increased hepatocellular necrosis or death. The observed microscopic hepatic effects were interpreted as reversible.In the 1000 mg/kg/day male rats the microscopic liver effects observed were associated with increased absolute and relative liver weights. However, in the female rat livers of all dose levels were affected, despite the absence of an increased liver weight in this sex. The microscopic hepatic fatty change was noted in essentially all 1000 mg/kg/day males and all treated groups of females; because a low incidence was observed in controls of both sex, and low and middle dose males, it may represent normal variation associated with fasting. The microscopic vacuolization in the periportal hepatocytes seen in various groups of rats of both sexes may also be partially attributable to a variable degree of fasting prior to necropsy. The normal activity of coprophagy in the rat could also contribute to periportal hepatocellular vacuolization. Therefore, greater amounts of glycogen would still remain in the periportal hepatocytes. Whether the effects within the livers of male and female rats can be totally attributed to indirect nutritional effects is uncertain. It is likely that the response in the liver was due to nutritional considerations and the test chemical. The presence of the test chemical in their feed likely accentuated this effect in high dose males, and all dose group of females, with subsequent fatty change also being observed. An alternative explanation for the observed liver fatty change is related to the known potential of a number of aliphatic alcoholic amines to cause a deficiency in the nutrient choline in rats and mice. AMP has been shown to disrupt phospholipid synthesis (Cosmetic Ingredient Review, 1990). As reviewed by Zeisel et al. (1995), choline requirements of pregnant rats in particular are substantially increased as the developing fetus is dependent upon maternal supplies of choline. The latter would explain the observed sensitivity of pregnant females to the liver fatty change relative to males. AMP-induced choline deficiency may also have accounted for the increased incidence of fetal resorbtions in the present study consistent with findings of resorptions in mice fed choline-deficient diets.There were no histopathological lesions per se in the uterus. However, in light of the aforementioned effect on post-implantation loss, four sections of uterus were microscopically examined from each female rat to ascertain the presence of one or more implantation sites. In the control, low, and middle dose animals which delivered a litter, the uterus contained small numbers of individual pigment laden macophages within the deep layer of the endometrial stroma and the myometrium, with a large aggregation of these pigmented cells in the myometrium associated with the mesometrial attachment. This is the normal appearance of a post-partum uterus. In the remaining middle and high-dose females which had totally resorbed litters, the uteri did not have the typical microscopic findings of an implantation site usually located in the region of the myometrium associated with the mesometrial attachment. However, a clearly recognizable change was present in the endometrium likely associated with a previous implantation. Immediately beneath the uterine mucosa, within the endometrial stroma, there were increased numbers of pigment laden macrophages. In five high-dose females, the pregnancies were detected by microscopic examination of the uterus. Overall, the microscopic uterine findings in rats that did not litter were consistent with embryonic death and subsequent resorption. Normal estrous cyclic activity in all the control and all treated female rats was observed microscopically in the tissues examined. Microscopic findings in the tissues examined from the adults did not reveal a likely mechanism for the observed effects on post-implantation loss. However, the fatty changes in the liver of all dose levels of female rats and the 1000 mg/kg/day males does suggest a possible effect on normal lipid and triglyceride metabolism. Whether similar effects may have been present in tissues of the rapidly developing fetus or involved in the normal formation of the placenta to maintain pregnancy was not determined in this study. Certainly the normal metabolism of lipids and triglycerides is a critical function for cellular membrane synthesis, and could have affected fetal viability. Endocrine glands were not microscopically examined in this study to determine whether a morphologic change was present.Male reproductive organs and their accessory sex glands were unaffected by treatment. The microscopic observations in tissues other than liver and uterus, were not interpreted to be due to treatment with the test chemical.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
other: NOEL (toxicity)
Effect level:
300 mg/kg bw/day
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Generation not specified (migrated information)
Key result
Dose descriptor:
other: NOEL (toxicity)
Effect level:
< 100 mg/kg bw/day
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
other: NOEL (reproductive effects)
Effect level:
100 mg/kg bw/day
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Increased post-implantation loss at 300 mg/kg/day and higher.(210 mkd AMP)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean pup body weights in the 300 mg/kg/day group ((210 mkd AMP) were increased on days 1 and 4 postpartum, with the male mean pup weights identified as statistically different. These increases were most likely due to the decreased litter size at this dose level, as pup body weight varies inversely with litter size (Agnish and Keller, 1997). Mean pup body weights of dams given 1000 mg/kg/day (700 mkd AMP) could not be evaluated due to a lack of viable litters.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no visible external morphologic alterations noted in any of the offspring delivered. There were no treatment-related effects on mean pup body weights in the 100 mg/kg/day group (70 mkd AMP). Mean pup body weights in the 300 mg/kg/day (210 mkd AMP) group were increased on days 1 and 4 postpartum, with the male mean pup weights identified as statistically different. These increases were most likely due to the decreased litter size at this dose level, as pup body weight varies inversely with litter size (Agnish and Keller, 1997). Mean pup body weights of dams given 1000 mg/kg/day (700 mkd AMP) could not be evaluated due to a lack of viable litters.

Effect levels (F1)

Key result
Dose descriptor:
other: No effects observed
Generation:
F1
Effect level:
> 100 - < 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

No effects on mating or conception. Liver effects in females at 100 mg/kg/day (70 mg/kg/day AMP) and higher, increased post-implantation loss at 300 mg/kg/day (210 mg/kg/day AMP) and higher.

Applicant's summary and conclusion

Conclusions:
Dietary exposure of male rats to 1000 mg/kg/day of AMP HCl (700 mg/kg/day AMP) caused increases in absolute and relative liver weights, accompanied by a very slight degree of microvacuolization of periportal hepatocytes, with or without vacuolization of hepatocytes consistent with fatty change. Females in all treatment groups exhibited similar histopathological changes in the liver, but in the absence of an organ weight change. Absolute and relative kidney weights were increased in the 1000 mg/kg/day of AMP HCl in males (700 mkd AMP), but these were not considered toxicologically significant due to the absence of histopathological changes. AMP HCl had no effect on mating performance or conception, but caused marked, dose-related increases in post-implantation loss (embryo resorption). At the high dose level of of AMP HCl, all 12 pregnant females showed evidence of complete litter resorption (100% post-implantation loss), while at 300 mg/kg/day (210 mkd AMP), post-implantation loss was 70% (vs. 10% in controls). Effects associated with, or secondary to the post-implantation loss increase at 300 mg/kg/day (210 Mkd AMP) included decreased litter size, increased pup body weight, and decreased gestation body weight and body weight gain. There were no treatment related effects on reproductive performance in the 100 mg/kg/day group for AMP HCl (70 mkd AMP).The no-observed effect level (NOEL) for general toxicity in males was 300 mg/kg/day, (210 mkd for AMP) while the general toxicity NOEL for females could not be determined, based upon the presence of very slight microscopic liver effects. The NOEL for reproductive effects was considered to be 100 mg/kg/day (70 mkd AMP).