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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
Not specified in report
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: CRL:CD(SD)
Details on test animals 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

open allclose all
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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.