Reaction product of alkylarylsulphonic acid and cycloaliphatic amine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In the 2 generation study with 4,4-dimethyl oxazolidine parental animals (P1) showed ulceration and inflammation in the nasal region as well as stomach irritation (thickened wall of the non-glandular stomach) at 60 and 200 mg/kg bw. These effects were related to the release of formaldhyde from the substance. At 200 mg/kg bw animals were heavier with increased liver weights associated with altered staining and vacuolisaton. In addition an increased kidney weight was observed without any histopathogical findings. At 60 mg/kg bw an increased liver weight was seen in males only. Similar effects were found in P2 animals with again stomach irritation and/or vacuolisation of the liver cells as most pronounced effects.

No effects on reproduction except an increased post-implantations loss were found in P1 and P2 females at 200 mg/kg bw (the similar effect in P1 females at 60 mg/kg bw did not reach statistical significance). There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, time to mating, gestation length, or pup sex ratio in either generation. 

In both the F1 and F2 offspring no effects on body weight, organ weights or macroscopic effects were noted.

The NOAEL for parental animals is 20 mg/kg bw, for reproductive effects 60 mg/kg bw and for effects on development of the pups 200 mg/kg bw.

In a study according to OECD 422 with AMP, dietary exposure of male rats to 1000 mg/kg/day of 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 males, but these were not considered toxicologically significant due to the absence of histopathological changes. AMP 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, all 12 pregnant females showed evidence of complete litter resorption (100% post-implantation loss), while at 300 mg/kg/day, 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 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. The no-observed adverse effect level (NOAEL) for general toxicity in males was 300 mg/kg/day, while the general toxicity NOAEL for females could not be determined, based upon the presence of very slight microscopic liver effects. The NOAEL for reproductive effects was considered to be 100 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental dates: 11/17/2006 to 8/3/2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC No. 0378-6978

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Sprague-Dawley rats (27/sex/dose) were obtained from Charles River Laboratories INC (Portage, MI, USA), and were 6 weeks of age at study initiation. Rats were acclimated to the testing facitility prior to random assignment to dose groups, and were offered feed and water ad libitum. They were housed in rooms designed to maintain adequate environmental conditions for the species (air changes, photocycle, temperature, and relative humidity).

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Groups of 27 male and 27 female Crl:CD(SD) rats were administered the test material seven days/week via oral gavage at dose levels of 0, 20, 60, or 200 mg CS-1135/kg of body weight/day (in polyethylene glycol 400 vehicle) for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations.

Details on mating procedure:

Breeding of the P1 adults commencee after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating 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 was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the male and returned to their home cage. If a breeding male died or was removed from study, a substitute partner (from the same dose group) that has already completed mating was provided, if available. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. Approximately 10 weeks after all F1 litters were weaned, F1 offspring were randomly selected to become P2 adults and were bred as described above. Cohabitation of male and female littermates was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation of CS-1135 in polyethylene glycol -400 (PEG) analyzed using gas chromatography with electron impact mass spectrometry detection (GC/EI/MSD) and quantified using isotopically labeled internal standard. Dose confirmation within +/- 14% of nominal concentrations

Duration of treatment / exposure:

10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations. F1 pups were exposed indirectly through milk and by gavage beginning at weaning.

Frequency of treatment:

once daily, 7-days/week

Remarks:

Doses / Concentrations:0, 20, 60, 200 mk/kg/dayBasis:nominal conc.

No. of animals per sex per dose:

27/sex/group

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Cage-side examinations were conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Any animals found dead were necropsied as soon as practical. Cage-side examinations were be conducted on dams and their litters, at least twice daily.Clinical examinations were conducted on 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 21. Clinical observations were not conducted on females that failed to mate or deliver a litter, unless deemed appropriate based on cageside observations. 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.All rats were weighed during the pre-exposure period and weekly during the 10-week pre-breeding periods. Males were weighed weekly after breeding until termination. Mated females were weighed on GD 0, 7, 14, 17, and 21. Lactating females were weighed on LD 1, 4, 7, 14, and 21. Females that failed to mate and/or deliver a litter were weighed weekly during the subsequent gestation and/or lactation segments of the study. F1 offspring assigned to the P2 generation that begin oral gavage dosing on PND 22 were weighed approximately every three days until they reached six weeks of age. The additional body weights collected were used for dose calculations to accommodate for rapid growth during this time.Feed consumption was determined weekly during the 10-week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured due to co-housing. Following breeding, feed consumption for males continued to be measured weekly until termination. For mated females, feed consumption was measured on GD 0, 7, 14, and 21. For females delivering litters, feed consumption was measured on LD 1, 4, 7, 11, 14, 17, 19, and 21. Feed consumption was not measured for females that failed to mate or failed to deliver a litter.

Oestrous cyclicity (parental animals):

Vaginal lavage samples from all P1 and P2 females were collected daily for three weeks prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period had elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring lavage fluid to a microscope slide. Vaginal lavage slides collected during the three weeks prior to mating were examined microscopically to determine estrous cycle length and pattern. On the day of scheduled necropsy, vaginal lavage samples were collected from all P1 and P2 female rats for subsequent determination of the stage of the estrous cycle.

Sperm parameters (parental animals):

Sperm parameters were evaluated in all P1 and P2 males at termination. Unless circumstances dictated otherwise, the left and right epididymides and testes were allocated as follows: right epididymis - motility and histopathology; left epididymis - counts; right testis - histopathology; left testis - counts.MotilityImmediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing SpermPrep Medium (ZDL, Lexington, Kentucky) and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. Images from the motility analyses were recorded on CD-R and were archived with the study file. After sperm were released, the epididymis was placed in Bouin's fixative and subjected to histologic examination.CountsThe left testis and cauda epididymis were weighed and then frozen at approximately -20 C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count determined from an aliquot loaded into the IVOS analyzer as described by Stradler et al. (1996). Initially, samples from the high-dose and control animals were evaluated. If treatment-related effects were seen, the middle and low-dose groups will also be evaluated as necessary to establish a NOEL.MorphologyAn aliquot of sperm suspension was placed on a slide, and a smear prepared and air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm from control and high-dose males was conducted. If treatment-related effects were observed, evaluation of sperm from the lower dose levels was performed. Sperm morphology was scored blind with respect to treatment group.

Litter observations:

Females were observed periodically 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 parturition was recorded as the first day that one or more delivered fetuses are noted, and was designated as LD 0. The following information was recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any pup found dead or sacrificed in moribund condition was sexed and examined grossly, to the extent possible, for external and visceral defects. Cage-side examinations were be conducted on litters, at least twice daily.To minimize variation in pup growth due to differences in litter size, all litters were standardized to eight pups per litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were randomly selected using a computer generated randomization procedure, so that four males and four females remained in each litter. If it was not possible to have four pups/sex in each litter, unequal numbers of males and females were retained (e.g., five males, three females). Litters with fewer than eight pups were not culled. Preferential culling of runts was not performed. Culled pups were euthanized and then discarded. All litters were weaned on PND 21. All F1 weanlings selected for mating, were observed daily for vaginal opening beginning on PND 28 (Cooper et al., 1989) or preputial separation beginning on day 35 (Korenbrot et al., 1977). The age and body weight at the time of landmark acquisition were recorded. Examination for puberty onset ceased upon acquisition, or on PND 43 (females) or 62 (males), whichever came first.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all adult animals. The necropsy included an examination of the external tissues and all orifices. Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) and known target organs will be recorded, and the organ-to-body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters. Histologic examination of the tissues (Table A6.8.2/01-1) was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose, and relevant gross lesions and reproductive organs of animals with signs of reduced fertility. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 µm and stained. The presence and integrity of the 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 defines 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, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P2 females in the control and high-dose groups, 15 per group were randomly selected for this examination.Selected histopathologic findings wiere graded to reflect the severity of specific lesions

Postmortem examinations (offspring):

Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning will be submitted on PND 22 for a complete necropsy. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues and any known target organs were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter were randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated.

Statistics:

Parental body weights, gestation and lactation body weight gains, litter mean body weights, feed consumption, anogenital distance (absolute and relative to the cubed root of body weight), sperm count, follicle count, percent total and progressively motile sperm, mean estrous cycle length and organ weights (absolute and relative) will be evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) will be performed. If the ANOVA is significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) will be performed. Feed consumption values will be excluded from analysis if the feed is spilled or scratched.Gestation length, age at vaginal opening (females), age at preputial separation (males), average time to mating, and litter size will be analyzed using a nonparametric ANOVA. If the ANOVA is significant, the Wilcoxon Rank-Sum test with Bonferroni's correction will be performed. Sperm morphology will be arcsine transformed and analyzed using a parametric ANOVA. Slides containing less than 200 sperm will be excluded from analysis. If the ANOVA is significant, the Dunnett's test will be performed. Statistical outliers (alpha = 0.02) will be identified by the sequential method of Grubbs (1969) and will only be excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices will be analyzed by the Fisher exact probability test

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:Female mating index = (No. females with evidence of mating/No. paired) x 100Male mating index = (No. males with evidence of mating/No. paired) x 100Female conception index = (No. females with evidence of pregnancy/No. mated) x 100Male conception index = (No. males siring a litter/No. mated) x 100Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100Male fertility index = (No. males siring a litter/No. paired) x 100Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100Gestation survival index = percentage of delivered pups alive at birthPost-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100

Offspring viability indices:

The following information was recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21.Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100Day 7, 14, or 21 pup survival index = (No. viable pups on day 7, 14 or 21/No. live after culling) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Gastric irritation due to release of formaldehyde

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological 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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)Treatment-related clinical observations in the P1 animals included one high-dose male that was euthanized in moribund condition. Necropsy revealed marked stomach ulceration and inflammation in the nasal region as contributors to the moribund condition of this rat. Gastric irritation due to release of formaldehyde from the test material likely caused this treatment-related effect.TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)Analyses of dose solutions for concentration verification were conducted on four different occasions during the study, representing all dose levels, sexes and major study phases. The mean concentrations of CS-1135 in the vehicle for all analyses ranged from 85.8 to 105.9% of the targeted concentrations. The overall mean concentration for the entire study ranged from 92.3 to 95.9% of the targeted concentrations. Analyses also confirmed that the test material was homogeneously distributed in the vehicle with relative standard deviations ranging from 0.5 to 4.7%.ORGAN WEIGHTS (PARENTAL ANIMALS)The absolute and relative liver weights of P1 males and females given 200 mg/kg/day were heavier than the controls, statistically identified, outside of the historical control range and were interpreted to be treatment-related. These higher liver weights were associated with microscopic altered staining of hepatocytes and vacuolization consistent with fatty change in males and females given 200 mg/kg/day. The absolute and relative liver weights of P1 males given 60 mg/kg/day were outside of the historical control data, but were not statistically identified. These higher liver weights were accompanied by treatment-related microscopic liver effects and were interpreted to be treatment-related. The liver weights of females given 20 or 60 mg/kg/day were not affected by treatment. The absolute and relative kidney weights of P1 males given 200 mg/kg/day were higher than the controls. Although only the increase in absolute kidney weight was statistically identified, both the absolute and relative kidney weights were outside of the historical control range and were interpreted to be treatment-related. These higher kidney weights were not associated with any microscopic treatment-related effects in the kidneys and may reflect an adaptive change associated with the excretion of the test material in the urine.GROSS PATHOLOGY (PARENTAL ANIMALS)The only organ affected by treatment was the nonglandular stomach. The nonglandular stomach was thickened in the majority of males and females given 200 mg/kg/day. In addition, a few females in this group also had an erosion or ulcer in the glandular mucosa. These treatment-related effects were confined to rats in the 200 mg/kg/day group and were suggestive of an adaptive response to irritation associated with the presence of formaldehyde as a degradation product of the test material. As was observed in the P1 adults, the only organ affected by treatment in the P2 adults was the stomach. The nonglandular stomach was thickened in the majority of the males and females given 200 mg/kg/day. Two males and one female in this dose group also had erosions or ulcers in glandular mucosa of the stomach.HISTOPATHOLOGY (PARENTAL ANIMALS)The primary treatment-related effect was observed in the stomachs of males and females given 200 mg/kg/day, and to a lesser extent in females given 60 mg/kg/day. These stomach effects were likely due to point of contact irritation, associated with the presence of formaldehyde as a degradation product of the test material. In the nonglandular stomach the effects were primarily characterized by hyperkeratosis and hyperplasia of the mucosa. In females the nonglandular effects were more focal and primarily affected the limiting ridge of the stomach. In addition, this minimal effect in the limiting ridge also affected several more rats in the 60 mg/kg/day rats than was observed in the low dose or control groups. This portion of the stomach is normally lined by squamous epithelium and this effect was interpreted to be an adaptive response to an irritant material. This was the region of the stomach with the grossly recognizable changes noted during necropsy. The glandular stomachs of P1 males and females given 200 mg/kg/day were also significantly affected in a number of animals and interpreted to be reflective of contact with an irritant material. Hyperplasia of the glandular mucosa of the stomach was evident in the majority of P1 males and females given 200 mg/kg/day, with the effect being slightly more severe in females than males. In addition, some of the rats given 200 mg/kg/day had one or more mucosal erosions and ulcers with an associated inflammation and edema in the mucosa or submucosa of the glandular region of the stomach. In the 60 mg/kg/day group, a single female also had a mucosal erosion with edema in the submucosa of the glandular stomach. This rat also had acute, focal, glandular inflammation in the mucosa. Several more females given 60 mg/kg/day had subacute to chronic inflammation in the glandular submucosa, that was either focal or multifocal, and very slight in degree, when compared to the incidences in the control or 20 mg/kg/day group. The erosions, ulcers, edema, hyperplasia and inflammatory infiltrates primarily involved the fundic portion of the glandular stomach. The microscopic findings in the liver of males and females given 60 or 200 mg/kg/day were considered treatment-related. The microscopic findings were characterized by 1) altered tinctorial properties; increased eosinophilia; hepatocyte; centrilobular of a very slight or slight degree; and 2) vacuolization consistent with fatty change, hepatocyte; individual cells; multifocal, very slight. All of the other microscopic findings in the remaining tissues were spontaneous occurrences or associated with a gavage incident. The primary treatment-related effect was observed in the stomachs of P2 males and females at 200 mg/kg/day. As in the P1 generation, these stomach effects were likely due to point of contact irritation, associated with the presence of formaldehyde as a degradation product of the test material. In contrast to the minimal effect observed in the P1 60 mg/kg/day female stomach, this dose group was not affected in the P2 group. The responses in nonglandular and glandular portions of the stomach were identical to those in the P1 males and females with only slight numerical differences. The microscopic findings in the liver which appeared to be treatment-related were the same in P2 males and females as in P1 rats. Vacuolization and altered tinctorial properties of the hepatocytes were the same. The changes in both were apparent in males at 60 and 200 mg/kg/day. Several more males had a slight grade of vacuolization in these groups in contrast to a very slight grade in P1 males. In the P1 females there were fewer control and 20 mg/kg/day rats with vacuolization of hepatocytes in contrast to the P2 groups. Therefore, the liver in P2 60 mg/kg/day females was unaffected. In the male P2 livers there was a slight increase in the number of rats with multifocal, and slight degree of aggregates of macrophages as histocytes. The increased kidney weights and adrenal weights were not associated with any microscopic findings due to the test chemical. All of the other microscopic findings observed in other tissues were considered normal spontaneous events or associated with changes secondary to gavage accidents. Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effect of treatmentThere were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females given 200 mg/kg/day as compared to the control females.OTHER FINDINGS (PARENTAL ANIMALS)There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, time to mating, gestation length, or pup sex ratio in either generation.There was a statistically identified increase in percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) in the mid- and high-dose group of the P1 litter, and the high-dose group of the P2 litter. The increase in postimplantation loss was considered treatment-related for the P1 and P2 high-dose litters because of the statistical significance and because the values were outside of the historical control ranges. The statistically identified increase in postimplantation loss of the mid-dose P1 litter was considered to be a spurious finding unrelated to treatment because the value was within the historical control range, was not present in the P2 litter at this dose level, and was largely driven by one dam with clear signs of maternal toxicity.

Key result

Dose descriptor:

NOEL

Remarks:

Systemic Toxicity

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Remarks:

Reproductive Toxicity

Effect level:

60 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Due to slighlty increased post-implantation loss at 200 mg/kg/day

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

Ophthalmological findings:

not examined

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):

Liver weight increases in males at treatment levels 60 and 200 mg/kg/day

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolization and altered tinctorial properties of the heptaocytes in males at 60 and 200 mg/kg/day. Similar results were seen in P1 females at the highest dose level. Effects similar to those seen in stomachs of P0 males and females were seen at the highest dose level

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

Systemis toxicity

Effect level:

20 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Vacuolization and altered tinctorial of hepatocytes

Key result

Dose descriptor:

NOEL

Remarks:

Reproductive toxicity

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Due to slightly increased post-implantation losses at 200 mg/kg bw/day

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

VIABILITY (OFFSPRING)There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, time to mating, gestation length, or pup sex ratio in either generation. In the P1 generation, there was a slight, but not-statistically identified, decrease in gestation survival index in the 200 and 60 mg/kg/day dose groups (96.0 and 95.5%, respectively) compared to controls (99.7%). Animals in the 200 mg/kg/day dose group also had slight, but not-statistically identified, decreases in PND 1 and 4 survival indices (96.5 and 94.8%) compared to controls (99.5 and 98.7). Because these values were not statistically significant, were near or just slightly outside the historical control range, and were not reproduced in the second generation, they were deemed spurious and unrelated to treatment.There was a statistically identified increase in percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) in the mid- and high-dose group of the P1 litter, and the high-dose group of the P2 litter. The increase in postimplantation loss was considered treatment-related for the P1 and P2 high-dose litters because of the statistical significance and because the values were outside of the historical control ranges. The statistically identified increase in postimplantation loss of the mid-dose P1 litter was considered to be a spurious finding unrelated to treatment because the value was within the historical control range, was not present in the P2 litter at this dose level, and was largely driven by one dam (6158, see Clinical Observations section) with clear signs of maternal toxicity.ORGAN WEIGHTS (OFFSPRING)There were no treatment-related alterations in organ weights of F1 or F2 weanlings at any dose level. See "Observations: parental animals" for results on organ weghts of adult P2 animalsGROSS PATHOLOGY (OFFSPRING)There were no treatment-related gross pathologic observations. See "Observations: parental animals" for results on gross pathology of adult P2 animalsHISTOPATHOLOGY (OFFSPRING)See "Observations: parental animals" for results on gross pathology of adult P2 animals.

Key result

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

There were no effects on any parameter of reporductive performance or offspring growth and survival at 20 or 60 mg/kg/day

Key result

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Generation:

F2

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system 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.

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. 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

Remarks:

Doses / Concentrations:0, 100, 300, 1000 mg/kg/dayBasis:nominal in diet

No. of animals per sex per dose:

12/sex/dose

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. 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

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.

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

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: 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)

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.

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)

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights in the 300 mg/kg/day 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 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

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

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. Mean pup body weights in the 300 mg/kg/day 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 could not be evaluated due to a lack of viable litters.

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

Reproductive effects observed:

not specified

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

Conclusions:

Dietary exposure of male rats to 1000 mg/kg/day of 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 males, but these were not considered toxicologically significant due to the absence of histopathological changes. AMP 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, all 12 pregnant females showed evidence of complete litter resorption (100% post-implantation loss), while at 300 mg/kg/day, 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 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.The no-observed effect level (NOEL) for general toxicity in males was 300 mg/kg/day, 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The data on the degradation product AMP confirm the findings with 4,4-dimethyl oxazolidine. In absence of data on PTSA the data for 4,4-dimethyl oxazolidine will be used in a worst case

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In a developmental toxicity on 4,4-dimethyl oxazolidine with rabbits gastric irritation and decreased body weight gain in maternal animals at 40 mg/kg bw was observed during day 7 to 28 of gestation. There was also a treatment-related, statistically identified increase in para-ovarian cysts (variation) in the fetuses of dams from this group. This finding was deemed to be of no toxicological significance due to the high prevalence in adult rabbits, the lack of any apparent parenchymal involvement, and the absence of any accompanying developmental effects. There were no treatment-related maternal or developmental effects any other dose groups. The NOAEL for maternal toxicity is 12 mg/kg bw and for developmental toxicity 40 mg/kg bw (the highest dose tested)d

In a dose range finding study on developmental toxicity study with rats, all animals given 750 mg/kg/day or higher died between GD 6 and GD 9. One animal given 500 mg/kg/day died on GD 14. Decreased dose-related mean body weight (gain) occurred during the initial 3 days of dosing in the 250 and 500 mg/kg groups, but thereafter body weight gain was comparable to the control group at 250 mg/kg and was slightly decreased at 500 mg/kg. Necropsy findings of the stomach were the most prevalent animals that died (reddened or thickened stomach mucosa) In survivors, there were three incidences of dilated renal pelvis (one at 250 mg/kg, and two at 500 mg/kg). One animal at 250 mg/kg had a distended ureter. Mean numbers of viable fetuses, early and late resorptions, implantation sites, and corpora lutea in animals given 250 and 500 mg/kgy were comparable to the controls. There was no indication of embryotoxicity at dose levels of 250 or 500 mg/kg/day

The maternal NOAEL is 250 mg/kg bw. The NOAEL for developmental effects is 500 mg/kg bw.

 

Dermal administration of 300 mg/kg/day of AMP (HCl salt) produced significant effects at the test site, as evidenced by scabbing (77% affected) and moderate to severe scaling (35% affected). The dermal finding of slight scaling at 30 and 100 mg/kg/day was not considered adverse, as the observation was transient in nature and relatively low in incidence. There was no evidence of test article related systemic maternal or developmental toxicity at any dose level tested. Under the conditions of this study, the NOAEL for maternal toxicity based on dermal effects was 100 mg/kg/day. The NOEL for developmental toxicity was 300 mg/kg/day, the highest dose level tested. Analyses of blood samples confirmed systemic exposure to AMP in a dose-responsive manner, although the study was not designed to quantify percent absorption.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 May 2008 to 17 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Covance Research Products (CRP), Inc. (Kalamazoo, Michigan)- Age at study initiation: five to six months - Weight at study initiation: 2500-3500g - Fasting period before study: None - Housing: Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle). Cages had flattened tube grid floors and were suspended above catch pans with absorbent non-contact bedding. Cages contained a J-type feeder and a pressure activated lixit valve-type watering system. There was also a variety of stainless steel objects attached to the front of the cages for environmental enrichment. - Diet: 8 oz (~180 g) LabDiet Certified Rabbit Diet #5325 (PMI Nutrition International, St. Louis, Missouri) - Water: ad libitum- Acclimation period: 6-7 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20°C ± 1°C - Humidity (%): 40-60 - Air changes (per hr): 12-15 times/hour - Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)IN-LIFE DATES: From: 12 May 2008 To: 17 June 2008

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

Groups of 26 time-mated rabbits will be orally gavaged 7 days/week on days 7-27 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CS-1135 solutions were prepared in a vehicle of PEG 400 and administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. Dose solutions were prepared periodically based on stability data.Dose confirmation analyses of all dose levels, plus control, were conducted on the first mix prior to administration. The homogeneity of the low-dose and the high-dose solutions was determined concurrent with dose confirmation. The method for analyzing the test material in PEG 400 was solvent dilution of dose solutions followed by analysis using gas chromatography-mass spectrometry (GC/MS) and quantitation using an internal standard method. CS-1135 was shown to be stable in PEG 400 at concentrations ranging from 2.5–500 mg/mL for at least 33 days. At a concentration of 0.25 mg/mL, CS-1135 was shown to be stable in PEG 400 for at least 14 days (Mielke, 2006). Dose solutions for the current study were prepared and used within these stability limits.

Details on mating procedure:

time-mated by supplier

Duration of treatment / exposure:

days 7-27 of gestation

Frequency of treatment:

once daily

Duration of test:

gestation days 7-27

Dose / conc.:

4 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Maternal examinations:

Cage side and clinical observations were conducted at least daily. Body weights were recorded on GD 0 by the supplier, daily during the dosing period, and on GD 28. Statistical analysis of body weights were performed using data collected on GD 0, 7, 10, 13, 16, 20, 24, and 28. Statistical analysis of body weight gains were conducted for the following intervals: GD 0-7, 7-10, 10-13, 13-16, 16-20, 20-24, 24-28, 7-28, and 0-28. Daily feed consumption was recorded and statistically analyzed for all animals from GD 4-28.

Ovaries and uterine content:

On GD 28, all surviving adult females (not fasted) were submitted for a complete necropsy. The liver and kidneys were weighed and organ:body weight ratios calculated. A detailed examination of the reproductive tract was performed and the number and position of implantations, viable 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. 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 and examined for evidence of early resorptions in order to verify pregnancy status. For females with one or more viable fetuses, the number of ovarian corpora lutea were counted.

Fetal examinations:

All fetuses were weighed, and 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 then euthanized by sublingual oral administration of sodium pentobarbital solution. All fetuses were also given a visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. 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 fetuses were sexed by examination of the gonads.Approximately one half of the fetuses in each litter were randomly selected for craniofacial examination. 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. All fetuses were then eviscerated, preserved in alcohol and stained with Alizarin Red S in order to visualize ossified bone. After staining, skeletons were macerated and cleared, and a thorough evaluation of the fetal skeleton was conducted.All fetal alterations will be 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 will be conducted such that investigators are blind to treatment group assignment.

Statistics:

Maternal BW, maternal BW gain, organ weight (absolute and relative), fetal BW and feed consumption were evaluated by Bartlett's test (a=0.01) for equality of variances. Based on the outcome of Bartlett's test, a parametric or nonparametric ANOVA was performed. If the ANOVA is significant at a=0.05, analysis by Dunnett's test (a=0.05) or the Wilcoxon Rank-Sum test (a=0.05) with Bonferroni's correction was performed, respectively. Feed consumption values were excluded from analysis if the feed is spilled or scratched. Frequency of pre- and post- implantation loss, and fetal alterations were analyzed using a censored Wilcoxon test with Bonferroni's correction. The number of corpora lutea, implantations, litter size were evaluated using a nonparametric ANOVA (a=0.05) followed by the Wilcoxon Rank-Sum test (a=0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (a=0.05) with Bonferroni's correction. Fetal sex ratios were analyzed using a binomial distribution test. Females lacking visible implantations were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (a=0.02), and if excluded, were excluded for sound scientific reasons. Both Dunnett's test and Bonferroni's correction correct for multiple comparisons to the control group to keep the experiment-wise a=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) was 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 are consistent with other biological and pathological findings and historical control values.

Indices:

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

Clinical signs:

effects observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Non significantly different from controls

Ophthalmological findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Deemed to be of limited toxicological significance

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Incidence not statistically significant

Total litter losses by resorption:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Details on maternal toxic effects: mean body weights for all treated groups were not significantly different from controls throughout the duration of the study. However, animals in the 40 mg/kg/day dose group had a clear, treatment-related, 19.3% decrease in mean body weight gain from GD 7-28, relative to controls. This effect was largely attributable to a 79% decrease in body weight gain from GD 24-28, which correlated with decreased food consumption during this period and was partly driven by body weight losses in two animals (1906 and 1916). There were no treatment-related effects on body weight gain in the 4 or 12 mg/kg/day dose groups. In the 40 mg/kg/day dose group, feed consumption during the last week of gestation, tended to be lower than controls, with the decreases being statistically identified GD 25-28. This decrease was concomitant with body weight loss and/or decreased body weight gain during this period.

Key result

Dose descriptor:

NOEL

Remarks:

Maternal toxicity

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

not specified

Description (incidence and severity):

Animals in 40 mg/kg/day group had treatment-related 19.3% decrease in mean body weight from GD 7-28 relative to controls.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yesDetails on embryotoxic / teratogenic effects:There was also a treatment-related, statistically identified increase in paraovarian cysts (variation) in the fetuses of dams from this group. This finding was deemed to be of no toxicological significance due to the high prevalence in adult rabbits, the lack of any apparent parenchymal involvement, and the absence of any accompanying developmental effects. There were no treatment-related maternal or developmental effects any dose group.

Key result

Dose descriptor:

NOEL

Remarks:

Maternal toxicity

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

other: Maternal toxicity based on decrease body weight gain, decreased feed consumption, and increased watey contents of the cecum.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other: No toxicologically significant findings at the highest dose level tested

Key result

Dose descriptor:

LOEL

Remarks:

Developmental toxicity

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other:

Remarks:

Statistically identified increase in paraovarian cysts (variation) in the fetuses of dams from the 40 mg/kg/day group

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: female reproductive system

Description (incidence and severity):

Treatment-related, statistically indentified increase in paraovarian cysts in the fetuses of dams dosed at 40 mg/kg/day. This effect was deemed to be of no toxicological significance due to high prevalence in adult rabbits, the lack of parenchymal involvement and the absence of developemental effects

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

Based on these findings the maternal no-observed-effect-level (NOEL) was considered to be 12 mg/kg/day, while the developmental toxicity no-observed-adverse-effect-level (NOAEL) was 40 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental dates: 6/4/1988 to 6/26/1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: dose-range finding study, meets generally accepted scientific standards and is described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

Dose range-finding study for definitive OECD 414

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Females weighed >200 g in order to be assigned to the study. Housed in suspended wire-mesh cages. Purina Certified Rodent Chow #5002 aand water vailable ad libitum. Temperature 69 to 73F, humidity 48-70%. 12-h light:dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

orally gavaged 7 days/week on days gestation days 6-15, inclusive

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

A sexually mature virgin female, was naturally mated with one male of the same strain. Positive evidence of mating was confirmed by the presence of a copulatory plug. Each mating set was examined daily. The day on which evidence of mating was identified was termed day 0 of gestation and the animals were separated.

Duration of treatment / exposure:

gestation days 6-15

Frequency of treatment:

once daily

Duration of test:

20 days

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Maternal examinations:

All animals were observed twice daily during gestation for effects on appearance and behaviour. Body weights were recorded at days 0, 6, 9, 12, 16, and 20. All survivors were sacrificed on gestation day 20 for a scheduled uterine examination.

Ovaries and uterine content:

The number of corpora lutea were counted on each ovary. The uterus as examined and the number and location of fetuses, early and late resorptions, and the total number of implantation sites were recorded in utero. Uteri with no macroscopic evidence of nidation were excised, opened, and placed in ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

no fetal examinations

Details on maternal toxic effects:

Maternal toxic effects:yesDetails on maternal toxic effects:All animals given 750 mg/kg/day or higher died between GD 6 and GD 9. One animal given 500 mg/kg/day died on GD 14. Prior to death, the following observations were noted: tremors, prostration, lethargy, rales, shallow respiration, paleness, salivation, decreased body temperature, lacrimation, and dried perioral soiling. There were no adverse signs of toxicity noted in animals at 250 mg/kg/day.Dose-related mean body weight losses and decreased body weight gains occurred during the initial 3 days of dosing in the 250 and 500 mg/kg/day groups. Body weight gain following the treatment period (GD 16-20) was comparable to the control group at the 250 mg/kg/day dose level and was slightly decreased at the 500 mg/kg/day dose. Total mortality by GD 9 at dose levels of 750 mg/kg/day and higher precluded further assessment of body weight data.Necropsy findings of the stomach were the most prevalent in victims; reddened or thickened stomach mucosa was the most frequent, followed by, less frequently, gaseous stomach, reddened and enlarged adrenals, reddened lungs, reddened medullary area in the kidney, clear or gelatinous material in the stomach and hemorrhagic thymus. In survivors, there were three incidences of dilated renal pelvis (one at 250 mg/kg/day, and two at 500 mg/kg/day). One animal in the 250 mg/kg/day group had a distended ureter.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

effects observed, treatment-related

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:Mean numbers of viable fetuses, early and late resorptions, implantation sites, and corpora lutea in animals given 250 and 500 mg/kg/day were comparable to the controls. Total maternal mortality in higher dose groups precluded further assessment of uterine data. There was no indication of embryotoxicity at dose levels of 250 or 500 mg/kg/day

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 APRIL 2004-05JANUARY 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

Not specified in report

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: CRL:CD(SD)

Details on test animals or test system and environmental conditions:

Time-mated female rats were obtained from a commercial supplier. Sexually mature adult rats were 10-11 weeks of age and weighed approximately 200-250 grams at study start.Each animal was evaluated by the laboratory veterinarian to determine the general health status and acceptability for study purposes upon arrival at the laboratory. Rats were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle) and acclimated for at least three days. Prior to dosing animals were acclimated to the wrapping material for approximately 2, 4, and 6 hours on GD 3, 4, and 5, respectively.Animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate environmental conditions for the species. Animals were provided feed and municipal water ad libitum. Rats 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. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Route of administration:

dermal

Vehicle:

other: deionized water

Details on exposure:

Rats were administered AMP once daily by the dermal (occluded) route at dose levels of 0, 30, 100, or 300 mg/kg/day for approximately 6 hours from GD 6-20. Dose solutions were prepared in deionized water at concentrations of 30, 100, and 300 mg/ml and pH adjusted to approximately 9.5 with HCl. The solutions were administered at a dose volume of 1 ml/kg body weight in order to achieve the targeted dose levels. Dose volumes were adjusted daily based on individual body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were prepared weekly during the course of the study. Homogeneity and stability were determined prior to study initiation, and concentration of the dose suspensions was determined from the first mix.

Details on mating procedure:

Sexually mature, adult virgin females were naturally mated with males of the same strain at the supplier's facility. 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 the testing laboratory on GD 1 or 2.

Duration of treatment / exposure:

GD6-20

Frequency of treatment:

6 hours/day

Duration of test:

15 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26 time-mated female rats/dose

Control animals:

yes, concurrent vehicle

Details on study design:

The maternal and developmental toxicity potential of 2-amino-2-methylpropanol (AMP) was evaluated. Groups of 26 time-mated female CD rats were administered AMP in deionized water (pH 9.5) by the dermal route at targeted dose levels of 0, 30, 100, or 300 mg/kg/day from gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On GD 21, all rats were euthanized and examined for gross pathologic 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. Blood samples were collected on GD 20 from four presumed pregnant females per group to evaluate blood levels of AMP and confirm its dermal absorption.

Maternal examinations:

A cage-side examination was conducted twice daily, at approximately the same time each day to detect significant clinical abnormalities that were clearly visible upon a limited examination, and used to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Following removal of the wrapping material at the end of each daily six-hour exposure period, the test sites were graded for erythema, edema, scaling, and fissuring. Body weights were recorded on GD 0 by the supplier, and daily during the dosing period, and at necropsy (GD 21). Feed consumption were measured and statistically analyzed for the following intervals: GD 3-6, 6-9, 9-12, 12-15, 15-18, and 18-21. On GD 21, all surviving females (not fasted) were euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was 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 stomach, liver, and kidneys 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 sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Toxicokinetic SubgroupThe first four presumed pregnant females from each dose group were selected for blood collection to evaluate systemic exposure following dermal administration of AMP. On the last day of dosing (GD20), following approximately three hours of dermal exposure, the designated animals were anesthetized using isoflurane and blood was collected from the orbital sinus. Approximately 0.5 ml of blood from each animal was collected into heparinized tubes. The samples were submitted to the analytical chemistry department for analysis and were stored frozen at approximately -80 C. AMP present in the blood was derivatized with pentafluorobenzoyl chloride under basic conditions and extracted in toluene. Quantitation was performed utilizing an isotopically labeled internal standard (D6-AMP) and matrix standards prepared in control blood. The limit of quantitation was 15 ng/g blood AMP.

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" would indicate a very recent death as evidenced by a lack 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 (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

Fetal examinations:

The sex of all fetuses was determined and the body weight of all viable fetuses 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. At least 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. 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. 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. After staining, skeletons were macerated and cleared. A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination. 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 fetal examinations were conducted such that investigators were blind to treatment group assignment.

Statistics:

see below

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

effects observed, treatment-related

Description (incidence and severity):

Dermal irritation at high dose

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Number of abortions:

no effects observed

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 pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yesDetails on maternal toxic effects: Dermal grading of the test site revealed a pattern of localized effects most pronounced in the 300 mg/kg/day dose group. The 300 mg/kg/day females showed significant dermal effects during the grading period, as compared to the other treatment groups. One 300 mg/kg/day female was found with very slight edema from GD 9-13. This finding was not observed in any other dose group. Almost all of the 300 mg/kg/day females (92%) were observed with slight scaling, with nine animals (36%) progressing to moderate to severe scaling during the last half of the dosing period. One, two, and seven females at 0, 30, and 100 mg/kg/day, respectively, were also found with slight scaling, but this was not considered adverse, as the incidence was relatively low, as compared to the 300 mg/kg/day group and the finding resolved prior to necropsy in three of the seven 100 mg/kg/day animals. In addition, no animals at the 30 and 100 mg/kg/day groups showed any signs of moderate to severe scaling. Scabbing was mainly observed at the 300 mg/kg/day dose level. Twenty 300 mg/kg/day females (77%) were noted with scabs at up to 25% of the test site. Over time, these the scabs progressed to include ever increasing areas of the test site, with one animal showing scabs covering most of the test site for the last week of dosing. The initial occurrence of scabbing varied considerably among the females, but there was a tendency for animals showing the greatest amount of scabs to have developed these lesions earlier. With the exception of one control animal, no other females were observed with any scabbing. Overall, the dermal grading procedures revealed a treatment-related effect at the high-dose level of 300 mg/kg/day. The slight, transient scaling seen at lower dose levels was not considered to be toxicologically significant.

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other:

Remarks:

Based on maternal dermal effects

Key result

Dose descriptor:

NOEL

Remarks:

Developmental toxicity

Effect level:

300 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Remarks on result:

other:

Remarks:

No effects at the highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): 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

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Remarks on result:

other:

Remarks:

No effects seen at highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: Maternal toxicity

Remarks on result:

other:

Remarks:

Dermal irritation at highest dose level tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Details on Maternal Results

Examinations performed on all animals during the course of the study revealed no treatment-related clinical findings. Dermal grading of the test site revealed a pattern of localized effects most pronounced in the 300 mg/kg/day dose group. The 300 mg/kg/day females showed significant dermal effects during the grading period, as compared to the other treatment groups. One 300 mg/kg/day female was found with very slight edema from GD 9-13. This finding was not observed in any other dose group. Almost all of the 300 mg/kg/day females (92%) were observed with slight scaling, with nine animals (36%) progressing to moderate to severe scaling during the last half of the dosing period. One, two, and seven females at 0, 30, and 100 mg/kg/day, respectively, were also found with slight scaling, but this was not considered adverse, as the incidence was relatively low, as compared to the 300 mg/kg/day group and the finding resolved prior to necropsy in three of the seven 100 mg/kg/day animals. In addition, no animals at the 30 and 100 mg/kg/day groups showed any signs of moderate to severe scaling. Scabbing was mainly observed at the 300 mg/kg/day dose level. Twenty 300 mg/kg/day females (77%) were noted with scabs at up to 25% of the test site. Over time, these the scabs progressed to include ever increasing areas of the test site, with one animal showing scabs covering most of the test site for the last week of dosing. The initial occurrence of scabbing varied considerably among the females, but there was a tendency for animals showing the greatest amount of scabs to have developed these lesions earlier. With the exception of one control animal, no other females were observed with any scabbing.

Overall, the dermal grading procedures revealed a treatment-related effect at the high-dose level of 300 mg/kg/day. The slight, transient scaling seen at lower dose levels was not considered to be toxicologically significant.

There were no statistically identified differences in the mean gestation body weight for females at any dose level.

Feed consumption was increased at 100 mg/kg/day during GD 3-9 interval. These statistically identified parameters were not considered treatment related, but attributed to relatively low control values.

There were no statistically identified differences in mean terminal body weight, mean liver and kidney weights, or relative liver and kidney to body weight ratios in the treatment groups, as compared to the control. Gross pathological findings were spurious in nature and not attributed to treatment.

There were no significant treatment related effects on pregnancy rate, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, fetal sex ratios, fetal body weights, or gravid uterine weights at any dose level. Mean percent postimplantation loss was statistically decreased in the 300 mg/kg/day group, but this was not considered to be test article-related, as the value was lower than seen in the control group. One female in the 30 mg/kg/day group was found to be non-pregnant, as confirmed by ammonium sulfide staining.

Toxicokinetic Subgroup

Blood samples were obtained from four presumed pregnant females on the last day of dosing following approximately three hours of dermal application. The results indicate dermal absorption of AMP in a dose-responsive manner. The disproportionately higher mean blood concentration of AMP at 300 mg/kg/day may have been the result of a compromised skin barrier, as evidenced by significant dermal effects (scabbing and scaling) at the test site for that group. At this dose level, one female (animal # 7169) was noted with a blood value approximately three times that of the others. The increase in the blood value for this animal is likely the result of a greater degree of compromise to the skin barrier, as evidenced by a greater degree of scabbing (up to 50%) at the test site on the day of blood collection, as compared to the other animals in the group.

Details on Embryotoxic/Teratogenic Results

There were no statistically significant differences in the incidence of any fetal alteration (malformation or variation) in any of the treated groups, as compared to the control. There were no external fetal alternations observed. Any reported findings were sporadic in nature, and were not considered toxicologically relevant or treatment related.

Conclusions:

Dermal administration of 300 mg/kg/day of AMP produced significant effects at the test site, as evidenced by scabbing (77% affected) and moderate to severe scaling (35% affected). The dermal finding of slight scaling at 30 and 100 mg/kg/day was not considered adverse, as the observation was transient in nature and relatively low in incidence. There was no evidence of test article related systemic maternal or developmental toxicity at any dose level tested. Under the conditions of this study, the NOAEL for maternal toxicity based on dermal effects was 100 mg/kg/day. The NOEL for developmental toxicity was 300 mg/kg/day, the highest dose level tested. Analyses of blood samples confirmed systemic exposure to AMP in a dose-responsive manner, although the study was not designed to quantify percent absorption.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The data on the degradation product AMP confirm the findings with 4,4-dimethyl oxazolidine. In absence of data on PTSA the data for 4,4-dimethyl oxazolidine will be used in a worst case

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The data on the degradation product AMP confirm the findings with 4,4-dimethyl oxazolidine. In absence of data on PTSA the data for 4,4-dimethyl oxazolidine will be used in a worst case