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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 May 2015 - 3 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Viscous liquid
Details on test material:
- Name of test material (as cited in study report): 2-(dimethylamino)-2-methyl-1-propanol (DMAMP)
- CAS nr. 7005-47-2
- Analytical purity: 99.15%.
- Lot number: 201205994-51

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
Test animals:
- Source: Charles River (Raleigh, North Carolina)
- Age and weight at study initiation: Sexually mature adult weighing approximately 200-250 g
- Fasting period before study: none
- Diet: LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form
- Water: municipal water was provided ad libitum (water was analyzed periodically for chemical parameters and biological contaminants)
- Acclimation period: 4 days
- Housing: after assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
- Temperature: 22°C with a range of 20°C-26°C
- Humidity: 50% with a range of 30-70%
- Air Changes: 10-15 times/hour (average)
- Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Ultrapure water
Details on exposure:
All dosing solutions were prepared by mixing the test material in Ultrapure water adjusted to approximately a final pH of 9, at concentrations of 0, 25, 75, or 250 mg/ml and administered at a dose volume of 4 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. The control rats were dosed with Ultrapure water adjusted to approximately a final pH 9 at 4 ml/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration verification of all dose solutions and homogeneity of the low- and high-dose solutions were determined pre-exposure. The method used for analyzing the test material in Ultrapure water was high performance liquid chromatography with positive ion electrospray ionization and tandem mass spectrometry detection operating in the multiple reaction monitoring mode (HPLC/ESI-MS/MS) (LCMS.
Details on mating procedure:
Sexually mature, adult virgin females were naturally mated with males of the same strain (one male:one female) at the suppliers 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 laboratory on GD 1 or 2.
Duration of treatment / exposure:
Days 6 - 20 of gestation
Frequency of treatment:
Once daily
Duration of test:
Gestation days: 6 - 21
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
24
Control animals:
yes
Details on study design:
Dose levels for this study were selected on the basis of the reproduction/developmental toxicityscreening study. Gavage exposure to DMAMP at doses up to 1000 mg/kg/day resulted in no treatment-related effects in clinical observations, body weights and body weight gains, feed consumption, reproductive function, prenatal/early neonatal growth and survival of the offspring, or gross pathology in either sex. The high-dose of 1000 mg/kg/day represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3700 Prenatal Developmental Toxicity Study). The lower dose levels were selected to provide dose response data for any toxicity that was observed among the high-dose group rats and to establish a no-observed-effect level (NOEL).

Examinations

Maternal examinations:
A cage-side examination was conducted twice daily, approximately at the same time each day. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. All animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Ovaries and uterine content:
On GD 21, all surviving females (not fasted) were sedated with a mixture of isoflurane vapors and medical oxygen, 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 skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. 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 portions of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of the liver, kidneys, and gross lesions was not conducted. Transponders were removed and placed in with the preserved tissues.

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” indicated a very recent death as evidenced by a lack of external degenerative changes. For females with one or more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations was stained with a 10% aqueous solution of sodium sulfide based on (Kopf et al., 1964) and examined for evidence of early resorptions in order to verify pregnancy status.

The sex and body weight of all viable fetuses were recorded. All fetuses were given an external examination that included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations (Staples, 1974; Stuckhardt and Poppe, 1984). The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue (Wilson, 1965). The remaining fetuses not selected for visceral examination were then skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone according to methods based on Trueman et al. (1999) and Zablotny (2002). A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination. However, a fetus may have been intentionally changed from one selected for visceral examination to one processed for skeletal examination (and vice versa) if it was deemed that such examination would have provided more meaningful data about a suspected abnormality.

All fetal alterations were classified as a variation or malformation. A variation is defined as a divergence beyond the normal range of structural constitution that may not adversely affect survival or health. A malformation is defined as a permanent structural change that may adversely affect survival, development or function and/or which occurs at a relatively low incidence in the specific species/strain. The maternal necropsy and fetal examinations were conducted such that investigators were blind to treatment group assignment.
Fetal examinations:
NOTE: all references to tables can be found in the original report.

The incidence of external, craniofacial, visceral and skeletal variations and malformations observed among fetal rats is summarized in Table 10, while individual litter data are reported in Appendix Table 8. Compared to controls, there were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups. The small number of alterations observed in fetuses from dams administered DMAMP either occurred at low frequencies and/or were not dose related. Details of these findings are described below.

External Examination
There were no treatment-related external alternations in any dose group. Incidental findings bearing no relationship to treatment included the e malformations aglossia, microagnathia, and gastroschisis umbilicus (Text Table 7). Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Craniofacial Examination
There were no craniofacial alternations in any dose group.

Visceral Examination
There were no treatment-related visceral alternations in any dose group. Incidental findings bearing no relationship to treatment included the malformations situs inversus, misshapen heart, and persistent 4th aortic arch (Text Table 8) and the variations fused lung, missing caudal lung lobe, hemorrhage liver, pale liver, supernumerary hepatic liver lobule, hemorrhage adrenal, bifurcated renal vein, and accessory blood vessel kidney. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.

Skeletal Examination
There were no treatment-related skeletal alternations in any dose group. In the 100 and 300 mg/kg/day dose groups there was a statistically -identified decrease in the incidence of delayed ossification (DO) of the thoracic centra compared to control (Text Table 9). This finding bore no toxicological significance as the incidence was lower than controls. Other incidental findings bearing no relationship to treatment included the malformation missing sternebrae (Text Table 10) and the variations supernumerary bone skull, DO parietal, DO occipital, DO thoracic centra, DO sternebrae, irregular pattern of ossification sternebrae, calloused ribs, class I and II wavy ribs, DO lumbar centra, and extra 1st lumbar rib. Given that these observations occurred in the control group, at low frequencies, and/or lacked a dose response, these observations were considered spurious and unrelated to treatment.
Statistics:
Maternal body weights, maternal body weight gains, maternal organ weights (absolute and relative), gravid uterine weights, fetal body weights, and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with
Bonferroni's correction (Miller, 1966) was performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations and litter size were evaluated using a non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios
were analyzed using a binomial distribution test. Females lacking visible implantations or totally resorbed litters at the scheduled necropsy were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha = 0.02; Grubbs, 1969) and, if excluded, were excluded for sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
See effects levels above.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
See effects levels above.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No-Observed-Effect Level (NOEL) is 300 mg/kg bw /day for both maternal and embryo/fetal toxicity. Based on these findings, it is not appropriate to classify this substance as a reproductive toxicant.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of 2-(dimethylamino)-2-methyl-1-propanol (DMAMP) in Crl:CD(SD) rats following repeated gavage administration. Groups of 24 time-mated female rats were administered DMAMP in Ultrapure water by gavage at dose levels of 0, 100, 300, or 1000 mg/kg/day on 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.

Maternal toxicity was limited to dams given 1000 mg/kg/day and consisted of treatmentrelated decreases in body weight, body weight gain and feed consumption during the treatment period compared to controls. There were no effects on body weight, body weight gain, or feed consumption in the 100 and 300 mg/kg/day dose groups. At necropsy there were no treatment-related gross pathological observations, and there were no treatment-related differences in kidney or liver weights for any of the treated groups when compared to their respective controls.

There were no treatment-related fetal malformations in any group up to, and including, 1000 mg/kg/day, the highest dose tested. In the 1000 mg/kg/day dose group there was a treatment-related decrease in fetal body weights (male 6.8%, female 5.8%, and combined 6.6%) compared to controls. There were no effects on fetal body weights in the 100 and 300 mg/kg/day dose groups.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 300 mg/kg/day, and the embryo/fetal NOEL was 300 mg/kg/day.