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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Two phases (Phase I, teratologic evaluation (similar to OECD 414); Phase II, postnatal evaluation) were embedded within each of two study replicates.
There were 6 consecutive breeding days in each replicate and 15 days between the last breeding day for Replicate I and the first breeding day for Replicate II.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Boric acid (CAS 10043-35-3) was previously determined by NTP to be 99% pure by inductively coupled plasma emission spectrometry (ICP) and 98% pure by titration with 0.1 N sodium hydroxide. Prior to this study, chemical identity was verified by infrared spectroscopy, and purity was 101% relative to a frozen reference aliquot.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD) BR VAF/Plus Outbred Rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC, USA
- Age at study initiation: females: 8 weeks, males: approx. 11 weeks
- Weight at study initiation: females on GD0 219 - 296 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum, Punna Certified Ground Rodent Chow No. 5002, Ralston, Punna Co, St. Louis, MO, USA
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 - 24.8
- Humidity (%): 27.6 - 92. 3
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): Punna Certified Ground Rodent Chow No. 5002, Ralston, Punna Co, St. Louis, MO, USA.
- Storage temperature of food: not specified

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Boron in control feed was below the method quantitation limit of 0.00281% boric acid (< 28.1 µg boric acid/g feed). Dosed feed contained boron equivalent to 91 - 109% of the nominal boric acid concentrations. At 0.025 or 0.2% boric acid in feed was homogeneous [96.3% (rsd = 2.7%) or 93.8% (rsd = 1.2%) of the nominal concentration] and stable (7 days at room temperature or 65 days under refrigeration).
Deionized/filtered drinking water sampled at the beginning and end of each study replicate contained < 0.30 µg boric acid/mL of water.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: not specified
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy (GD 0)
Duration of treatment / exposure:
Days 0 - 20 post mating (phase I)
Days 0 - 20 post mating then on normal diet until termination on day 21 postpartum (phase II)
Groups of 28 - 32 females were used for both phase I and phase II. In phase I the dams were killed on Day 20 for detailed fetal examination. In phase II the dams were allowed to deliver and the pups reared to weaning and then killed for full visceral and skeletal examination as for phase I.
Frequency of treatment:
continuously
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0.025 other: % boric acid
Remarks:
phase I: 3.3 mg boron/kg bw/day
phase II: 3.3 mg boron/kg bw/day
Dose / conc.:
0.05 other: % boric acid
Remarks:
phase I: 6.4 mg boron/kg bw/day
phase II: 6.3 mg boron/kg bw/day
Dose / conc.:
0.075 other: % boric acid
Remarks:
phase I: 9.6 mg boron/kg bw/day
phase II: 9.8 mg boron/kg bw/day
Dose / conc.:
0.1 other: % boric acid
Remarks:
phase I: 13.3 mg boron/kg bw/day
phase II: 12.9 mg boron/kg bw/day
Dose / conc.:
0.2 other: % boric acid
Remarks:
phase I: 25.2 mg boron/kg bw/day
phase II: 25.4 mg boron/kg bw/day
No. of animals per sex per dose:
Phase I (developmental toxicity phase): 28 - 32 per group
Phase II (postnatal phase): 28 - 32 per group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected to meet the principle objectives of the study. Specifically the proposed study was designed to determine whether the reduction of foetal body weight at 0.001% boric acid was repeatable, whether the NOAEL for foetal weight reduction could be determined and whether the induction of foetal skeletal anomalies at 0.2% boric acid was repeatable, whether the NOAEL for skeletal effects could be determined and whether the incidence of skeletal anomalies changed during postnatal life in the control and boric acid-exposed groups.
The period of exposure for all dietary concentrations of boric acid was GD 0 to 20, the same as for a previous study and would therefore allow determination of whether the effects were repeatable and establish a NOAEL under the same conditions.

- Rationale for animal assignment: On GD 0, timed-mated females were assigned to dose groups and study phase by stratified randomisation so that the body weights did not differ among groups within either study phase.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were not specified.

DETAILED CLINICAL OBSERVATIONS: not specified

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 3, 6, 9, 12, 15, 18, 19, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 19, and 19 to 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus

OTHER: organ weights were determined from liver, right kidney, and uterus
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 resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: [half per litter]
Statistics:
Maternal and developmental end points from each study phase were analyzed separately, and the litter was the experimental unit for all developmental measures. Statistical procedures were based on SAS software. In conjunction with analysis of variance (ANOVA), Bartlett's test for homogeneity of variance (a = 0.001) was applied to all data, and an arcsine-square root transformation was performed on litter-derived percentage data. ANOVA determined the significance of dose effects, replicate effects and dose x replicate interactions (a = 0.05). Dunnett's test (one-tailed) and Williams' test were used to compare treated groups to the control group, except that a two-tailed Dunnett's test was used for maternal organ and body weight parameters, maternal food and water consumption, fetal or pup body weight, and percentage males/litter. A test for linear trend determined the significance of dose-response relationships. For nonparametric analyses, the chi square test for independence determined differences among treatment groups, the Cochran-Armitage test evaluated linear trends on proportional data, and Fisher's exact test (one-tailed) compared individual treated groups to controls (a = 0.05).
Indices:
no data
Historical control data:
no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Maternal body weight did not differ among groups during gestation or lactation, and weight gain was similarly unaffected (data not shown). Decreasing trends for maternal body weight or weight gain after GD 15 (data not shown), or for the gestational period as a whole (Table 1 in the attachment), appeared to be secondary to the slight reduction in gravid uterine weight at the high dose (Table 1 in the attachment). Thus, maternal corrected weight gain (Table 1) and postpartum body weight (data not shown) were not affected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Marginally detectable, transient decreases in maternal food intake were observed during the first 3 days of exposure. The maximum decrease was 11% at the high dose. Otherwise, treatment did not appear to affect maternal food intake (data not shown). Average daily intake of boric acid (mg/kg body weight/day) was calculated from maternal body weight, measured food intake, and nominal concentration of boric acid in feed. Boron intake was calculated as 17.5% of boric acid intake (Table 1 in the attachment).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Relative maternal water consumption (g/kg/day) was transiently elevated at 0.05, 0.075, and 0.1% boric acid from GD 15 to 18 and also at 0.2% boric acid from GD 15 to 20 (Phase I only; data not shown), but was otherwise affected.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute (g) or relative (% body weight) maternal liver weight did not differ among groups on GD 20 or PND 21.
Relative, but not absolute, maternal right kidney weight was elevated in the 0.2% treatment group on GD 20, but no effect was observed on PND 21 (Table 1 in the attachment). This observation is not considered adverse but incidental since no dose relationship was observed.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

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:
not examined
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
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The percentage of dams delivering after GD 22 increased slightly with boric acid exposure, but differences among groups were not statistically significant (Table 2 in the attachment). The range of delivery days (GD 21-23) was within the historical control range (Charles River, 1993).
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Pregnancy was confirmed in 25 - 30 dams (88 - 100%)/group/phase (Table 1 in the attachment).
On GD 20, the number of ovarian corpora lutea/dam, number of implantation sites/litter, percentage preimplantation loss/litter, and live litter size were equivalent among groups (Table 2 in the attachment). Likewise, the percentages of resorptions or late fetal deaths were not affected. Spurious decreases in the percentage of litters with at least one resorption were not dose-related. The control incidence of resorptions was higher than expected, but the incidence among boric acid-exposed groups was similar to historical data for this species and strain. During lactation, the number and percentage of pup deaths/litter exhibited increasing trends, but the number of implantation sites/litter, cumulative offspring mortality (i.e., percentage of implantation sites) and number of live pups/litter did not differ among groups (Table 2 in attachment). Thus, there was no definitive evidence for an adverse effect on offspring viability from conception through weaning.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
25.2 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
boron
Basis for effect level:
other: no maternal toxicity observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
On GD 20, fetal body weight was significantly decreased by 6 and 12% at 0.1 and 0.2% boric acid, respectively. Fetal body weight deficits on GD 20 did not persist into the postnatal period (Tables 2 and 7; Fig. 1 in the attachment).
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:
not specified
Changes in litter size and weights:
not examined
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
On GD 20 and PND 21, external malformations and variations occurred with low incidences (<= 0.5% fetuses or pups/group) which showed no apparent dose-response relationships (Tables 3 and 4 in the attachment).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
On GD 20, the percentage fetuses with skeletal malformations/litter showed a significant increasing trend, but the overall incidence of skeletal variations was not affected (Tables 3 and 5 in the attachment). Short rib XIII (classified as a malformation) and wavy rib (classified as a variation) were each significantly increased in the 0.1 and 0.2% boric acid groups relative to controls on GD 20 (Table 5 in the attachment). Short rib XIII occurred in 0, 0, 0.2, 0.7, 1.5, and 3.4% of fetuses from the control through high-dose groups, respectively, and wavy rib (or wavy rib cartilage) occurred in 0, 0.4, 0, 0.9, 2.1, and 10% of fetuses. A significant decreasing trend was found for rudimentary (but not full) extra rib on lumbar I (classified as a variation), and a slight, but not statistically significant, decrease in the incidence of this finding was noted in the high-dose group on GD 20 (Table 5 in the attachment). Thus, the incidence of extra rib on lumbar I (full and/or rudimentary) was 3.1, 1.3, 2.8, 0.9, 1.5, and 0.2% of fetuses (GD 20) for the control through high-dose groups, respectively.

On PND 21, the overall incidence of skeletal malformations, showed a significantly increased incidence in the low and high dose groups relative to the control group (Tables 4 and 5 in the attachment). The incidence of short rib was 0, 1.3, 0.5, 0.2, 0.9 and 3.9% of pups examined in the control through high-dose groups, respectively. Based upon the absence of a dose-response relationship for short rib XIII across the nearly 10-fold concentration range between the low and high dose, findings of short rib XIII at concentrations below 0.2% boric acid in the diet did not appear to be treatment-related. On PND 21, the incidence of wavy rib (a variation) was low and not dose related, i.e., 0, 0, 0.3, 0.2, 0, and 0% in the control through high dose groups, respectively. On PND 21, there were no pups in the control or treated groups with extra rib on lumbar I (full or rudimentary) (Table 4 in the attachment).
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral malformations and variations also failed to show dose-related increases following boric acid exposure (Tables 3 and 4 in tha attachment). Among the visceral findings on GD 20 (Table 3 in the attachment), enlarged lateral ventricles, hydronephrosis, hydroureter, and distended ureter are relatively common findings for this species and strain in the testing laboratory.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
9.6 mg/kg bw/day (actual dose received)
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
incidence of short rib XIII and wavy ribs increased

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
13.3 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The teratogenicity of the test substance was assessed according to OECD guideline 414. There was no evidence of developmental toxicity in offspring of rats fed boric acid in diet throughout gestation up to 0.075% (9.6 mg boron/kg bw/day). At 0.1% boric acid (13.3 mg boron/kg bw/day) there was reduced fetal body weight, short and wavy ribs, and these effects disappeared during the postnatal period. Similar but more marked effects were observed at the highest dose of 0.2% (25.2 mg boron/kg bw/day) and apart from the short 13th rib, they also disappeared during the postnatal period.