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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:
N/A
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Developmental and teratogenicity study. Study was well conducted and documented. Three dose and one control groups were tested (25 rats/sex/group). GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SI0801.01 (alkyl dimethyl amine oxide)
- Molecular formula (if other than submission substance): N/A
- Molecular weight (if other than submission substance): 235.7
- Smiles notation (if other than submission substance): N/A
- InChl (if other than submission substance): N/A
- Structural formula attached as image file (if other than submission substance): see Fig.: N/A
- Substance type: Active
- Physical state: Liquid,
- Analytical purity: N/A
- Impurities (identity and concentrations): N/A
- Composition of test material, percentage of components: N/A
- Isomers composition: N/A
- Purity test date: N/A
- Lot/batch No.: N/A
- Expiration date of the lot/batch: N/A
- Radiochemical purity (if radiolabelling): N/A
- Specific activity (if radiolabelling): N/A
- Locations of the label (if radiolabelling): N/A
- Expiration date of radiochemical substance (if radiolabelling): N/A
- Stability under test conditions: N/A
- Storage condition of test material: N/A
- Other: N/A

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: Females: 65 days; Males: 42 days.
- Weight at study initiation: Females: 218 - 262 g; Males: 506 - 969 g.
- Fasting period before study: N/A
- Housing: Rats were individual housed except during cohabitation period. During cohabitation, each pair of male and female rats was housed in the male rat's cage.
- Diet (ad libitum): Certified Rodent Diet #5002 (Purina Nutrition International, St. Louis, Missouri) in individual feeders.
- Water (ad libitum): Local water that had been processed by passage through a reverse osmossis membrane (R.O. water) was available to the rats from an automatic watering system and/or individual water bottles. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: N/A


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26
- Humidity (%): 30 to 70
- Air changes (per hr): 10/hr
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: N/A To: N/A

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sterile Water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Solutions of the test substances were prepared weekly at the Testing Facility. Dosage calculations were adjusted for the 32% (w/v) concentration of the test substance. Prepared formulations were stored at room temperature and stirred continuously (magnetic stir plate with stir bar) during dosage administration.


DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: Room temperature.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Sterile Water
- Concentration in vehicle: N/A
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): N/A
- Purity: N/A
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
N/A
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: N/A
- Further matings after two unsuccessful attempts: N/A
- Verification of same strain and source of both sexes: N/A
- Proof of pregnancy: sperm in vaginal smear and/or a copulatory plug observed in situ will be referred to as day 0 of pregnancy
- Any other deviations from standard protocol: N/A
Duration of treatment / exposure:
Days 6 through 19 of presumed gestation.
Frequency of treatment:
Daily
Duration of test:
Approximately 4 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, and 200 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a dosage-range study (Angus Research Laboratories, Inc., Protocol 916-025P), in which dosage levels of 0, 32.5, 100, 325 and 650 mg/kg/day were evaluated.
Dosages of 325 and 650 mg/kg/day were excessively toxic to the dams and conceptuses. At the 32.5 and 100 mg/kg/day dosage levels, no developmental toxicity was observed. Excess salivation was observed at the 100 mg/kg/day dosage level, and slight, non-statistically significant decreases in maternal body weight gains and feed consumption values were observed at the 32.5 and 100 mg/kg/day dosage levels. Doses of 200, 100, and 25 mg akly dimethyl amine oxide/kg/day were chosen.
- Rationale for animal assignment (if not random): Unpon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Female rats were assigned to four dosage groups (Groups I through IV), twentry-five rats per dosage group, using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0.
- Other: N/A

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations checked were: mortality, moribundity, pertinent behavioral changes and other signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly and Rats were also examined for clinical observations immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimation, on DG 0, daily during the dosage period and on DG 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: N/A

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: N/A

OTHER: N/A
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: A late resorption was defined as one in which the occurrence of organogenesis was grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses are considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions are differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetuses was a late resorption.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Bionomial Distribution.
Continuous data (e.g., body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and feal anomaly data) were analyzed using Bartletts' Test of Homogeneity of Variances and the Analysis of Variance, when appropriated [i.e., Bartlett's Test was not significant (pCount data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
N/A
Historical control data:
N/A

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY:
Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.
A single rat in the 200 mg/kg/day dosage group was found dead on day 19 of gestation (DG 19) after 13 daily dosages. This dam lost body weight after DG 9 and had reduced feed consumption values throughout the study. Adverse clinical observations included excessive salivation (DGs 8 to 9, 14 and 17 to 18), gasping (DGs 9 to 11 and 17 to 18), labored breathing (DGs 10 to 13 and 16 to 18), ungroomed coat (DGs 10 to 13), brown perianal substances (DGs 12 to 13), urine-stained abdominal fur (DGs 12 to 14 and 16 to 18), brown perivaginal substance (DGs 12 to 15), chromorhinorrhea (DG 15), rales (DG 16) and emaciation (DGs 17 to 18). No gross lesions were revealed by necropsy; all tissues appeared normal for moderate degree autolysis. There were 17 early resorptions in utera. This death was attributed to effects of the test substance because similar observations occurred in surviving rats in this dosage group.
A single rat in the 200 mg/kg/day dosage group was found dead on DG 18 after 12 daily dosages. This death was attributed to an intubation accident. This dam lost body weight after DG 13 and had reduced feed consumption values throughout the study. Adverse clinical observations included rales (DGs 10 to 11 and 13), excessive salivation (DGs 13 to 14), gasping (DGs 13 to 14), labored breathing (DGs 13 to 17), red perioral substance (DGs 15 to 17), urine-stained abdominal fur (DGs 15 to 17), dehydration (DG 16) and emaciation (DG 17). External observations at necropsy included red substance on fur of the nose, forepaws and forelimbs. Gross necropsy revealed a tear in the esophagus; all other tissues appeared normal for slight degree of autolysis. There were 14 fetuses in utero. The viability of the fetuses could not be determined because of the maternal death.

CLINICAL OBSERVATIONS:
Adverse clinical observations occurred at increased incidences in the 100 and 200 mg/kg/day dosage group rats. Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased (pAll other clincal observations were considered unrelated to the test substance because: 1) the incidences were not dosage-dependent; and/or 2) the observations occurred in only one or two rats. These clinical observations included one 200 mg/kg/day dosage group dam with an axillary mass attributed to an intubation error, and localized alopecia (limbs and underside) in one or two rats in the 0 (Vehicle), 25, and 200 mg/kg/day dosage groups.

NECROPSY OBSERVATIONS:
All necropsy observations were considered unrelated to the test substance because they were single events. These observations included slight dilation of the pelvis of the right kidney of one vehicle group dam and one 200 mg/kg/day dosage group dam. A single dame also had a distended urinary bladder with ten calculi and thickened and red bladder walls. One 100 mg/kg/day dosage group dam had large adrenals, four dark red areas on the fundic mucosa and numerous raised tan areas on the pyloric mucosa of the stomach, the intestines and stomach were distended with gas and the left lateral lobe of the liver was mottled. One 200 mg/kg/day dosage dam had a mass in the left axilla in-life; at necropsy, a clear tan gelatinous fluid was located subcutaneously in the area of the ventral neck, left axilla and leateral chest, the esophagus had a thickened area and the plyoric folds of the stomach were thickened. These observation were presumed to be the sequelae of a presumed intubation error.

MATERNAL BODY WEIGHTS, GRAVID UTERINE WEIGHTS AND BODY WEIGHT CHANGES:
Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced (pThe gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced (pBody weights and body weights gains were unaffected at dosages of 25 mg/kg/day of the test substance. Gravid uterine weights were unaffected by the 25 and 100 mg/kg/day dosages of the test substance.

MATERNAL ABSOLUTE (g/day) and RELATIVE (g/kg/day) FEED CONSUMPTION VALUES:
Absolute (g/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced (p
CAESAREAN-SECTIONING:
Caesarean-sectioning observations were based on 24, 25, 24 and 22 pregnant rats in the 0 (vehicle), 25, 100 and 200 mg/kg/dosage groups, respectively. Male and female fetal body weights were significantly reduced (pNo other Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 200 mg/kg/day. The litter averages for corpora lutea, implantations, late resorptions, percent resorbed conceptuses (calculated excluding the completely resorbed litter in the 200 mg/kg/day dosage group), and percent live male fetuses were comparable among the four dosage groups and did not significantly differ. There were no dead fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
FETAL ALTERATIONS:
Fetal alterations were defined as: 1) malformations (irreversible changes that occur at low incidences in this species and strain); or 2) variations (common findings in this species and strain and reversible delays or accelerations in development). Litter averages were calculated for specific fetal ossification sites as part of the evaluation of the degree of fetal ossification.
Fetal evaluations were based on 339, 375, 348, and 297 Ceasarean-delivered live fetuses in 24, 25, 24, and 21 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. Each fetus was examined for gross external alterations, approximately one half of the fetuses in each litter were examined for soft tissue alterations and the remaining fetuses were examined for skeletal alterations and the number of ossification sites.
The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased (pAll other gross external, soft tissues or skeletal alterations (malformations or variations) were considered unrelated to the test substance because 1) the litter and fetal incidences were not dosage-dependent; 2) the alteration occurred in only one fetus; or 3) the incidences were within the ranges observed historically at the Test Facility.

FETAL ALTERATIONS:
In groups I through IV, litters with fetuses with alterations numbered 8 (33.3%), 11 (44.0%), 8 (33.3%), and 15 (71.4%), respectively. The numbers of fetuses with any alterations were 11 (3.2%), 25 (6.7%), 14 (4.0%) and 32 (10.8%) respectively. The percentages of fetuses with any alteration per litter were 3.6%, 6.4%, 4.5% and 11.5% in the four respective dosage groups.
The significant increases in the percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were considered to reflect delays in skeletal ossification, related to the significantly reduced (p
FETAL GROSS EXTERNAL ALTERATIONS:
One vehicle group fetus had whole body edema (anasarca); this fetus also had an absent innominate artery at soft tissue examination. One 25 mg/kg/day fetus had a thread-like tail; at skeletal evaluation, this fetus had fused arches of the 3rd sacral vertebra and no caudal vertebrae. One 200 mg/kg/day group fetus had a kinked tail, the 4th and 5th digits of the lift hindlimb were fused and a skin tab located to the left of its tail. At skeletal evaluation of this fetus, further evaluation of the skin tab revealed two bones (possibly a femur and fibula) fused together on the left side of the pelvis. An extra paw appeared to be attached to the underside of the left paw; also the centra of the 6th and 11th thoracic vertebrae were bifid. One 200 mg/kg/day fetus had a micrognathia; soft tissue evaluation of this fetus revealed a small tongue.

MALFORMATIONS - SOFT TISSUE EVALUATION:
One 200 mg/kg/day fetus had a small tongue; micrognathia was identified at gross external evaluation.

VARIATIONS - SOFT TISSUE EVALUATION:
One control group fetus and one 25 mg/kg/day dosage group fetus had an absent innominate vessel; whole body edema (anasarca) was identified at gross external evaluation for the control fetus, also an additional fetus had the umbilical artery descending to the left of the urinary bladder.

MALFORMATION - SKELETAL ALTERATIONS:
One control group fetus had fused arches of the 4th cervical vertebra; additional variations in ossification occurred in this fetus (unossified 1st sternal centra, incompletely ossified pubes).
One 25 mg/kg/day dosage group fetus had fused arches of the 3rd sacral vertebra; this fetus also had no caudal vertebrae (thread-like tail was noted at gross external examination).

VARIATIONS - SKELETAL ALTERATIONS:
A cervical rib was present at the 7th vertebra in one 25 mg/kg/day fetus. This fetus had no other skeletal alterations.
A bifid centrum in the thoracic vertebrae occurred in 1, 6, 5 and 9 fetuses from 1, 3, 5 and 8 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups respectively. One fetus in the 25 mg/kg/day dosage group also had incompletely ossified 1st and 2nd sternal centra, a fetus in the 100 mg/kg/day dosage group also had a bifid centrum of the 1st lumbar vertebra and a fetus in the 200 mg/kg/day dosage group also had incompletely ossified pubes.
An incompletely ossified arch in the 6th lumbar vetrebra occurred in one 100 mg/kg/day dosage group fetus; this fetus also had incompletely ossified ischia and pubes.
The significant increase (pDelayed sternal ossification (incompletely ossified and/or not ossified 1st and/or 2nd sternebrae) occurred in 4, 8, 3 and 11 fetuses from 4, 6, 2 and 7 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidence of only incompletely ossified 1st sternal centra was also significantly increased (pThe significant increases (pThe pubes and/or ischia were incompletely ossified in 8, 12, 7 and 16 fetuses from 6, 4, 3 and 6 litters in the 0 (vehicle), 25, 100 and 200 mg/kg/day dosage groups, respectively. The fetal incidenc of only incompletely ossified pubes was significantly increased (pThe significant increase (pThe litter averages for ossified caudal vertebrae, sternal centers and metacarpals per fetus were significantly decreased (pAnalyses of the average numbers of fetal ossification sites per fetus did not reveal any other statistically significant differences among the four dosage groups. Ossification of the hyoid, vertebrae (cervical, thoracic, lumbar, and sacral) ribs, sternum (manubrium and xiphoid), forelimbs (carpals and phalanges) and hindlimbs (tarsals, metatarsals and phalanges) occurred at similar incidences in litter in all dosage groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

N/A              

Applicant's summary and conclusion

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) for the test substance (alkyl dimethyl amine oxide) is 25 mg/kg/day. The 200 mg/kg/day dosage caused mortality in one dam, the 100 and 200 mg/kg/day dosages caused adverse clinical observations, reductions in body weight gain and reduced feed consumption values. Reductions in relative feed consumption values were also noted in the 25 mg/kg/day dose group; however, no concomitant adverse effects were noted at this dosage level. The developmental NOAEL was 25 mg/kg/day; the 200 mg/kg/day dosage caused reduced fetal body weights and associated delays in skeletal ossification and the 100 mg/kg/day dosage also caused delays in skeletal ossification.
Executive summary:

One-hundred Crl:CDBR VAF/Plus presumed pregnant female rats were randomly assigned to four dosage groups (Groups 1 through IV), 25 rats per group. The test substance preparations for dosing were corrected for purity. The test substance, was administered orally (via gavage) once daily to these female rats on days 6 through 19 of presumed gestation (DGs 6 through 19), at dosages of 0 (Vehicle), 25, 100, and 200 mg alkyl dimethyl amine oxide/kg/day. The dosage volume was 5 mL/kg, adjusted daily on the basis of the individual body weights recorded before intubation. The rats were intubated at approximately the same time each day.

The rats were observed for viability at least twice a day and for general appearance weekly during acclimation and on DG 0. The rats were also examined for clinical observations of effects of the test substance, abortions, premature deliveries and deaths immediately before and approximately 60 minutes after dosage and on the day of sacrifice (DG 20). Body weights were recorded weekly during acclimation, on DG 0, daily during the dosage period and on DG 20. Feed consumption values were recorded on DGs 0, 6, 9, 12, 15, 18, and 20.

All surviving rats were sacrificed on DG 20, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group in order to minimize bias. The number of corpora lutea in each ovary was recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantations, live and dead fetuses and early and late resorptions. The gravid uterus was weighed. Each fetus was removed from the uterus and subsequently weighed and examined for sex and gross external alterations. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations using a variation of the microdissection techniques. The remaining fetuses in each litter were examined for skeletal alterations.

Two rats in the 200 mg/kg/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice.

Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, labored breathing and gasping occurred in 100 mg/kg/day dosage group rats and in significantly increased numbers of 200 mg/kg/day dosage group rats. Additionally, chromorhinorrhea occurred in one and two rats in these two dosage groups, respectively. Observations of brown or red perivaginal substance, emaciation, brown perianal or perinasal substance, dehydration, ungroomed coat and soft or liquid feces occurred in one or two rats in the 200 mg/kg/day dosage group. All necropsy observations were considered unrelated to the test substance.

Rats in the 100 and 200 mg/kg/day dosage groups had significantly reduced body weight gains for the entire dosage period (calculated as days 6 to 20 of gestation) and body weight gains were significantly reduced in the 200 mg/kg/day dosage group for the entire gestation period (DGs 0 to 20). Body weights were significantly reduced in the 200 mg/kg/day dosage group on DGs 11 through 20. The gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced in the 200 mg/kg/day dosage group.

Absolute (g/day) and relative (g/kg/day) feed consumption values for the entire dosage period (calculated as DGs 6 to 20) and the entire gestation period (DGs 0 to 20) were significantly reduced in the 200 mg/kg/day dosage group. Relative feed consumption values for the entire dosage period were also significantly reduced in the 100 mg/kg/day dosage group, while values for the gestation period were significantly reduced in the 25, 100 and 200 mg/kg/day dosage groups.

Male and female fetal body weights were significantly reduced in the 200 mg/kg/day dosage group. Live litter size was decreased and the number of early resorptions was increased in the 200 mg/kg/day dosage group, but apparently as the result of one dam in this dosage group that had a litter consisting of 16 early resorptions.

The percentages of fetuses and litters with alterations in the 200 mg/kg/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced fetal body weights in this group. These delays in ossification included significant increases in the fetal and/or litter incidences of bifid thoracic vertebrae centra, incompletely and/or not ossified 1st or 2nd sternal centra, incompletely ossified pubes and significant decreases in the numbers of ossified caudal vertebrae, sternal centers and metacarpals. Additionally, delays in ossification occurred in the 100 mg/kg/day dosage group and included a significant increase in the litter incidence of bifid thoracic vertebrae centra.

NOAEL for both maternal toxicity and developmental toxicity is 25 mg/kg/day.