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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 23, 1998 - November 9, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Method: other: US EPA guidline (1997)
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): Sodium Chlorate
- Molecular formula (if other than submission substance): NaClO3
- Molecular weight (if other than submission substance): 106.44
- Physical state: a colorless, odorless crystal or white powder with a salty taste
- Analytical purity: Purity was determined by argentimetric titration and ion chromatography with suppressed conductivity detection. The overall purity was estimated as 99.2% (Battelle, March 24, 1998), Purity of the bulk chemical was verified as 99-100% (Battelle, October 26, 1998). Both documents are attached to the report (see last pages).
- Composition of test material, percentage of components: The identity was confirmed by infrared spectroscopy and elemental analysis of sodium and chlorine.
CoA ((Battelle, March 24, 1998): Sodium Chlorate (99.2%), Bromate (<0.015%), Chloride (<0.005%), Iron (<1ppm), Heavy metals (<0.0005%), Potassium (0.002%), Magnesium and Calcium precipitate (<0.005%), Nitrogen compounds (<0.0005%), Calcium, Magnesium&R203 precipitate (<0.005%), Sulfate (<0.001%)
- Purity test date: probably March 24, 1998 (Battelle)
- Lot/batch No.: 03623LR
- Expiration date of the lot/batch: No info
- Stability under test conditions: No info
- Storage condition of test material: No info
- Other: Obtained from Aldrich Chemical Company, Milwaukee, WI.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: no info
- Weight at study initiation: (P) Females: 3017 g - 3976 g (replicate I), 2747 g - 4061 g (replicate II)
- Fasting period before study: no info
- Housing: individually housed in stainless steel cages with mesh flooring
- Diet (e.g. ad libitum): Purina Certified Rabbit chow. From gestational day (gd) 3: food ad lib, (on gd 1: 65 g and on gd 2: 125 g)
- Water (e.g. ad libitum): tap water, ad lib
- Acclimation period: no info


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 20 ºC (replicate I and II)
- Humidity (%): 46.9 - 62.2% (replicate I), 49.0 - 60.7% (replicate II)
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12:12 light: dark cycle


IN-LIFE DATES: From: August 23, 1998 To: November 9, 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 0, 100, 250 and 475 mg/kg bw in deionized/distilled water
Sodium chlorate was dissolved in deionized/distilled water. Each concentration of sodium chlorate was formulated independently in a quantity sufficient to last the entire dosing period. Dose formulations were stored in sealed plastic botlles at room temperature, and mixed well prior to dosing. During the study, two sets of dose formulations were prepared (one set per replicate).

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (deionized/distilled water)
- Concentration in vehicle: 33.3, 83.3, 158.3 mg/ml
- Amount of vehicle (if gavage): total volume applied: 3 ml/kg
- Lot/batch no. (if required): not required
- Purity: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Formulations were administered within the period of proven stability (44 days).
- Aliquots were submitted for verification of concentration before each period of use. Measured concentrations were within 98.2%-102.1% of the theoretical concentration. (BLOQ = below the limit of quantitation. Limit of Quantitation = 0.11348 µg/ml).
- Nothing mentioned about homogenicity.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (animals were naturally mated at vendor’s facility prior to shipment).
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: no info, however, gd 0 = date of mating and pregnancy status was confirmed by uterine examination.
Duration of treatment / exposure:
gestation day 6 through 29
Frequency of treatment:
daily dose (1 oral dose per day. From day 6 through day 29 of gestation).
Duration of test:
Necropsy on gestation day 30.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 250 and 475 mg/kg bw d
Basis:
nominal conc.
No. of animals per sex per dose:
96 females, 24 per group, 12 per replicate
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: day 6 through day 30 of gestation
- Dose selection rationale: Doses were selected on the basis of a screening study in which New Zealand White Rabbits (8/group) were given 100, 250, 500, 750 or 1000 mg/kg bw via gavage from gd 6-29. At the two highest dose morbidity and mortality was seen (and these groups were terminated at gd 24). In the 500 mg/kg group one female died on gd 26 and lethargy and respiratory distress were seen in the other animals on gd 28-29. Clinical signs were minimal at dose <250 mg/kg d. Above 250 mg/kg d significant reduction of food uptake was seen in the exposed animals. Until the 500 mg/kg d dose overal weight changes during treatment and during gestation (corrected for gravid uterine weights) were not affected. There were no statistically significant differences among groups for gravid uterine weight, absolute or relative maternal liver weight. Pregnancy rates were 83-100% per group. A slight (not significant) decrease in number of corpora lutea, implantation sites and live fetuses per litter were seen >500 mg/kg d. Above 500 mg/kg d there was no definitive evidence of developmental toxicity. Therefore 475 mg/kg bw was chosen as the highest dose in the main study.
- Rationale for animal assignment (if not random): no info

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Females were observed for clinical condition at least once each day from 1 or 2 (date of animal arrival) through day 5 (prior to dosing). From day 6 through 29, females were observed for clinical condition and signs of toxicity at dosing, and approximately 1-2 hours after each dose. On day 30, females were observed for clinical condition at weighing and at scheduled termination.


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweight were recorded on day 0 (vendor), 3, 6 through 30 and after sacrifice on day 30.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule: Feed consumption was monitored during the study, with measurements on the mornings of day 3, 6, 9, 12, 15, 18, 21, 24, 27, 29, and 30.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: The body, liver, and gravid uterus of each female was weighed. Thoracic and abdominal cavities were examined. Sections of all livers, as well as any maternal organs which displayed gross pathology, were saved. Ovarian corpora lutea were counted. Pregnancy status was confirmed by uterine examination. Uterine contents were examined to determine the number of implantation sites, resorptions, dead fetuses, and live fetuses. Dead fetuses were counted, weighed, and discarded. Uteri which presented no visible implantation sites were stained in order to visualize any implantation sites which might have undergone very early resorption.


OTHER: At termination blood samples were collected from 2 females per group per replicate as well as from all animals killed before the end of the study. This blood was tested for pathogens.
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
Fetal examinations:
Fetuses: All live fetuses were counted, weighed, and examined for external morphological abnormalities, including cleft palate. All fetal carcasses were sexed and examined for visceral morphological abnormalities. Approximately 50% of the fetal carcasses were decapitated prior to dissection.
All fetal skeletons were examined for skeletal morphological abnormalities.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Unit for statistical measurement: pregnant female or the litter. Quantitative continuous data (e.g., maternal/fetal body weights, etc.) were evaluated for homogeneity of variance using Bartlett's test (Winer, 1962). (Reported when Bartlett's test indicated lack of homogeneity (p<0.001)).

Parametric statistical procedures were applied to selected measures from this developmental study. GLM procedures were applied to the ANOVA and the Tests for Linear Trend. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter derived percentage data. For litter-derived perc. data, the ANOVA was weighted according to litter size. When a significant (p<0.05) main effect for dose or replicate occurred, Dunnett's Multiple Comparison Test (Dunnett,1955;1964) was used to compare each treatment group to the control group for that measure. A one-tailed test (Dunnett's Test) was used for all pairwise comparisons to the control group, except that a two-tailed test was used for maternal body and organ weight parameters, maternal feed consumption, fetal body weight, and percent males per litter. Data for any measure which showed a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOVA was presented as Mean±SEM for each cell in the ANOVAdesign. Dose effects within each replicate were further evaluated using a one-way ANOVA, Test for Linear Trend, and Dunnett's Test. Nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, a one-tailed Fisher's Exact Probability Test (with adjustments for multiple comparisons) was used for pairwise comparisons between each treatment group and control group. Alpha level for each statist. comparison was 0.05, and significance levels for trend tests and pairwise comparisons were reported as p<0.05 or p<0.01.
Indices:
Particular focus on embryo/fetal growth, viability, and morphological development after implantation and prior to birth.
Historical control data:
Included in the report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Parental data and F1:
- Body weight: No significant treatment related effect was observed.
- Food/water consumption: No significant treatment related effect was observed on food consumption.
- Description, severity, time of onset and duration of clinical signs: urinary changes (orange or brown urine; little or no urine). The incidence of females exhibiting these signs was dose-related (i.e., 0, 0, 2 and 8 for color changes; 0, 1, 6 and 8 for reduced urine output). Urinary changes in individual females (one or both signs) were noted on one to seven days, but never for more than four consecutive days. Only two females (in the mid- and high dose group) showed both signs.
- Fertility index: -
- Precoital interval: -
- Duration of gestation: terminated at day 30.
- Gestation index: At termination of the study the pregnancy rates ranged from 90-100%.
- Changes in lactation: -
- Changes in estrus cycles: -
- Effects on sperm: -
- Hematological findings incidence and severity:
- Clinical biochemistry findings incidence and severity:
- Mortality: 1 animals died in each treatment group.
- Gross pathology incidence and severity:
Parents: Necropsy findings included primarily changes in lung appearance, which may have been secondary to gavage administration of the sodium chlorate. Gross necropsy findings did not occur in a dose-related manner.
- Number of implantations:
Dose 0 100 250 475
Sites 8.11+-/0.35 8.88+/-0.45 8.17+/-0.51 7.80+/-0.50
per litter
Not affected by treatment with sodium chlorate.
- Number of corpora lutea:
Dose 0 100 250 475
per dam 9.16+/-0.30 9.41+/-0.44 9.22+/-0.38 9.00+/-0.52
Not affected by treatment with sodium chlorate.
- Ovarian primordial follicle counts: -
- Organ weight changes: Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were similar among exposed and control animals.
- Histopathology incidence and severity: -
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 475 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 475 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:
- Offspring toxicity F1 and F2:
- Litter size and weights: Not affected by treatment with sodium chlorate.
- Sex and sex ratios: -
- Viability index: Not affected by treatment with sodium chlorate.
- Post natal survival until weaning: -
- Effects on offspring: -
- Postnatal growth, growth rate: -
- Vaginal opening (F) or preputial separation (M): -
- Gross pathology incidence and severity:
Fetuses: There were no effects of treatment with sodium chlorate on the incidence of external, visceral, or skeletal malformations at any dose.
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In summary, transient changes in maternal food intake, urinary color and/or output were noted at 100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998). Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were 475 mg/kg/day.
Executive summary:

The purpose of this study was to assess the effects of sodium chlorate on the pregnant female and on embryonic and fetal development when administered orally by gavage once daily to mated female New Zealand White rabbits from gestational days (gd) 6 through 29. Each group consisted of 24 mated female rabbits (12 per replicate). Sodium chlorate was administered once daily at dose levels of 0, 100, 250 and 475 mg/kg body weight/day. A standard dose volume of 3 mL/kg body weight was used. Control animals were dosed with the vehicle alone (deionized/distilled water). All surviving females were sacrificed on gestational day 30. Examination of dams and fetuses was performed in accordance with international recommendations. The study complies with OECD Guideline 414. Dose selection was based on a screening study in which New Zealand White rabbits were treated by gavage with 0, 100, 250, 500, 750, or 1000 mg sodium chlorate/kg body weight/day on gd 6 through 29. The study followed the principles of GLP.

Maternal toxicity:

Confirmed pregnancy rates were 90-100% per group. One maternal death occurred in each dose group. Clinical signs associated with sodium chlorate exposure included urinary changes (orange or brown urine; and/or little or no urine) on one or more days of treatment. The number of animals affected showed dose-response patterns across the control through high dose groups (i.e., 0, 0, 2, and 8 for color changes and 0, 1, 6 and 8 for urine output), respectively. Nevertheless, these changes in individual animals were transient and not clearly indicative of toxicity.

No significant treatment-related effects were observed for maternal body weight, body weight gain, or corrected weight gain. Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were equivalent among groups.

Relative maternal food intake (g/kg/day) exhibited an increasing trend prior to initiation of treatment, but was reduced to 82-83% of control intake in the mid and high dose groups during early treatment (gd 6 to 9). Thereafter, no significant differences were noted among treatment groups for maternal relative feed consumption, suggesting that the decrease on gd 6 to 9 was related to initiation of treatment. A decreasing dose-related trend for the periods of gd 15 to 18 and gd 18 to 21 was observed, although no significant differences between the control group and treated groups were observed for these periods. These effects were not noted later in the treatment period.

Fetal toxicity:

Sodium chlorate exposure did not significantly affect any endpoints related to prenatal viability. Average live litter size in sodium chlorate-treated groups was between 100-112% of the control mean, with no statistically significant difference among groups. There were no treatment-related effects on fetal body weight. There were no effects of treatment on the incidence of external, visceral, or skeletal malformations. In summary, transient changes in maternal food intake, urinary color and/or output were noted at ³100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998). Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were ³475 mg/kg/day.