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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:
19/11/1993 to 26/01/1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study. This study is conducted on an analogue substance. Read-across is justified on the following basis: In aqueous solutions at physiological and acidic pH, low concentrations of simple inorganic borates such as boric acid, disodium tetraborate decahydrate, disodium tetraborate pentahydrate, boric oxide and disodium octaborate tetrahydrate will predominantly exist as undissociated boric acid. At about pH 10 the metaborate anion (B(OH)4-) becomes the main species in solution (WHO, 1998). This leads to the conclusion that the main species in the plasma of mammals and in the environment is un-dissociated boric acid. Since other borates dissociate to form boric acid in aqueous solutions, they too can be considered to exist as un-dissociated boric acid under the same conditions. For comparative purposes, exposures to borates are often expressed in terms of boron (B) equivalents based on the fraction of boron in the source substance on a molecular weight basis. Some studies express dose in terms of B, whereas other studies express the dose in units of boric acid. Since the systemic effects and some of the local effects can be traced back to boric acid, results from one substance can be transferred to also evaluate the another substance on the basis of boron equivalents. Therefore data obtained from studies with these borates can be read across in the human health assessment for each individual substance. Conversion factors are given in the table below. Conversion factor for equivalent dose of B Boric acid H3BO3 0.175 Boric Oxide B2O3 0.311 Disodium tetraborate anhydrous Na2B4O7 0.215 Disodium tetraborate pentahydrate Na2B4O7•5H2O 0.148 Disodium tetraborate decahydrate Na2B4O7•10H2O 0.113 Disodium octaborate tetrahydrate Na2B8O13•4H2O 0.210 Sodium metaborate (anhydrous) NaBO2 0.1643 Sodium metaborate (dihydrate) NaBO2•2H2O 0.1062 Sodium metaborate (tetrahydrate) NaBO2•4H2O 0.0784 Sodium pentaborate (anhydrous) NaB5O8 0.2636 Sodium pentaborate (pentahydrate) NaB5O8∙5H2O 0.1832 References: WHO. Guidelines for drinking-water quality, Addendum to Volume 1, 1998.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Boric acid
- Purity test date: > 99 %
- Lot/batch No.: 872703 (Fisher Scientific Co.)
- Stability under test conditions: Stable

Test animals

Species:
rat
Strain:
other: Crl: CD VAF/Plus (Sprague Dawley)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, USA.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: 219 - 296 g
- Housing: Individually in solid-bottom polycarbonate cages with stainless steel wire lids

IN-LIFE DATES: From: 19/11/1993 To: 26/01/1994

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow. Within each of the two study replicates, each concentration of boric acid in feed was formulated independently in a quantity sufficient for use across the period of administration for both study phases within that replicate, except that two batch formulations were prepared at 0.025 % in Replicate 1.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of dosed feed by ICP indicated that the added concentrations of boric acid in feed were within 90.9 - 109.1 % of their nominal values. Boron levels in control rodent chow were less than 0.00281 % expressed as boric acid (i.e. less than 28.1 μg/g boric acid).
Mixes of the ground rodent chow were homogenous with the mean boric acid for 250 μg/g found at 96.3 % of nominal and a relative standard deviation of 2.7 %. For the 2000 μg/g dose level, the mean boric acid level found was 93.8 % of nominal with a relative standard deviation of 1.2 %.
The chow dosed with 250 μg/g boric acid was stable at room temperature over a period of 7 days with a recovery of 96.8, 100.4 and 100.7 % if nominal boric acid on days 0, 3 and 7 respectively. Similarly, after 7 days at room temperature, 20000 μg/g recoveries of 98.3, 96.3 and 93.7 % of nominal were found on days 0, 3, and 7 respectively. The same feed formulations were stable for 65 days when refrigerated.
Details on mating procedure:
Time-mated. Males and females were cohabited (1:1) overnight and mating was assessed by examination for sperm in the vaginal lavage of each cohabited female or the presence of a copulatory plug. The study was performed in two replicates with two study phases embedded in each replicate. There were 6 consecutive breeding dates in each replicate and the replicates were separated by 15 days between the last breeding date for replicate 1 and the first breeding date of replicate 2.
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:
Daily
Duration of test:
Days 0 - 20 post mating
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.025, 0.050, 0.075, 0.1 or 0.2 % (0, 250, 500, 750, 1000, 2000 ppm) equivalent to 19 (3.3), 36 (6.3), 55(9.6), 76 (13.3) and 143 (25) mg boric acid (mg B)/kg bw
Basis:
nominal in diet
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 was could be determined and whether the induction of foetal seketal 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 2, the same as for a previous study and would therefore allow determination of whether the effects were repeatable and establish and 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

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes

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: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Over 3-day periods


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
other - foetuses alive and dead. Empty uteri were stained with ammonium sulphide to look for implantation sites.
Fetal examinations:
- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data
Statistics:
Statistical procedures for selected experimental endpoints were based on SAS software. Except for nominal scale measures, data were reported as mean ± SEM. The litter was the experimental unit for all measures of developmental toxicity. An alpha level of 0.05 was applied to all group-wise, pair-wise or trend tests, except for Bartlett's Test or as otherwise noted below. Statistical results were reported by providing p values for ANOVA as p < 0.05 or p < 0.01 for pairwise tests; and as p < 0.05, < 0.01 or < 0.001 for trend tests in order to provide information equivalent to that obtained from the computer-generated output for these analyses.
Analyses for effects of boric acid were conducted within each study phase as follows. Except for nominal scale measurements, data was analysed by Bartlett's test for homogeneity of variance with an alpha level of 0.001. ANOVA was used to determine the significance of dose effects, replicate effects and dose x replicate interactions. When a significant (p < 0.05) main effect of dose occurred, Dunnett's test (one-tailed) and William's test were used to compare each boric acid-treated group to the vehicle control group for that measure, except that a two-tailed Dunnett's test was used for maternal organ and body weight parameters, maternal food and water consumption, foetal or pup body weight and percent males per litter. In three cases when the main effect for dose approached significance ) p = 0.0505 for maternal water intake of GD 18 to 19 in Phase I, and p = 0.0571 for absolute or relative maternal food intake on GD 0 to 3 in Phase II, the Dunnett's and Williams' tests were also applied. An arcsine-square root transformation was performed on all litter-derived percentage data prior to analysis by ANOVA. A test for linear trend was used to determine the significance of the dose-response relationship for parametric analyses.
Indices:
No data
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
No clinical effects or effects on bodyweight. There was an increase in relative kidney weight in the highest dose group in phase I of the study but not in Phase II. The authors concluded that there was little evidence of maternal toxicity at any of the doses tested (Phase I or 11).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
143 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
76 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
76 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
55 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day
Based on:
element
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
13.3 mg/kg bw/day
Based on:
element
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
13.3 mg/kg bw/day
Based on:
element
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
9.6 mg/kg bw/day
Based on:
element
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Developmental effects were found in foetuses from animals exposed to 76 mg/kg bw boric acid (13.3 mg B/kg bw) and above (Phase I) which are mainly associated with foetotoxic activity. Specifically, a reduction in the mean foetal bodyweight per litter (6 % compared to controls) was observed in Phase I foetuses at 13.3 mg B/kg bw. At this dose, skeletal changes which included an increased incidence of short rib XIII (considered a malformation by authors of this study but a variation by most workers) and an increased incidence of wavy rib (considered a variation) were also observed. At the high dose for Phase I animals of 143 mg/kg bw boric acid (25 mg B/kg bw), the bodyweight reductions and skeletal changes were more pronounced. The reduction in incidence in extra rib on Lumbar I (a variation) which was noted in the previous rat study was not statistically significant here due to the low incidence in control animals (3.2 % in controls in this study compared to 14 % in the study from Heindel et al, 1992). There was no evidence of any increase in external or visceral malformations in any treatment group. The authors concluded that the NOAELs for the prenatal and postnatal study phases (Phase I and Phase II) were 9.6 and 12.9 mg B/kg low/d, respectively.
The animals from the Phase II group which were killed on postnatal day 21 showed no reduction in pup bodyweight in any group at any time point compared to controls, which indicates full recovery in the offspring already by postnatal Day 0 from treatment-related bodyweight effects. The rib variations observed in the foetuses (wavy rib) from Phase I were not observed in any dose group in Phase II. Only at the highest dose in Phase II (25.3 mg B/kg bw) was an increased incidence of short rib XIII observed.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 9.6 mg/kg bw/day (nominal)
Based on:
element
Sex:
male/female
Basis for effect level:
other: The authors concluded that the NOAELs for the prenatal and postnatal study phases (Phase I and Phase II) were 9.6 and 12.9 mg B/kg bw/d, respectively.
Remarks on result:
other: prenatal study phase
Dose descriptor:
NOAEL
Effect level:
>= 12.9 mg/kg bw/day (nominal)
Based on:
element
Sex:
male/female
Basis for effect level:
other: The authors concluded that the NOAELs for the prenatal and postnatal study phases (Phase I and Phase II) were 9.6 and 12.9 mg B/kg bw/d, respectively.
Remarks on result:
other: postnatal study phase

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal effects Phase I and II:

Parameter

Control

0.025 dose

0.05 dose

0.075 dose

0.10 dose

0.20 dose

Number of dams examined

60

60

60

60

60

60

Clinical findings during application of test substance

none significant in any group

 

 

 

 

 

Mortality of dams (%)

0

0

0

0

0

0

Abortions

0

0

0

0

0

0

Body weight gain

no sig differences

 

 

 

 

 

Food consumption

no sig differences

 

 

 

 

 

Water consumption

if test substance is applied with drinking water

no sig differences

 

 

 

 

 

Pregnancies

pregnancy rate or %

56/60

56/60

55/60

58/60

55/60

55/60

Necropsy findings in dams dead before end of test

none

 

 

 

 

 

Litter response (Caesarian section data) Phase I:

 

Control

0.025

dose

0.05

dose

0.075

dose

0.10

dose

0.20

dose

Number of pregnancies

27

29

27

29

30

27

Corpora lutea           No. per dam

                                  ±SEM 

19.4

0.7

19.3

0.3

19.4

0.7

17.9

0.6

19.0

0.6

19.0

0.7

Implantations           No. per dam

                                   ± SEM

16.4

0.7

16.5

0.6

16.6

0.7

15.8

0.7

16.4

0.7

16.0

0.7

Resorptions               % per litter

                                   ±SEM

9.5

3.6

3.3

1.0

2.6

0.8

3.9

0.8

6.7

3.4

4.8

1.1

Total number of live foetuses

417

461

437

437

471

411

pre-implantation loss            %

                                               ±SEM

(%)           

13.8

4.1

13.5

2.7

13.2

3.5

12.8

3.7

11.5

4.0

14.1

4.1

post-implantation loss          %

                                               ±SEM

(%)

9.5

3.6

3.3

1.0

2.6

0.8

4.7

1.0

6.7

3.4

4.8

1.1

total number of litters

26

29

27

29

29

27

fetuses / litter

 

 

 

 

 

 

live fetuses / litter                 number

                                               ±SEM

ratio

16.0

0.4

15.9

0.6

16.2

0.7

15.1

0.7

16.2

0.6

15.2

0.7

Litters with one or more late foetal deaths

0

0

0

7

0

0

foetus weight (mean)              male

                                               female

                                               average

3.71

3.52

3.61

3.64

3.47

3.56

3.62

3.45

3.54

3.60

3.38

3.50

3.48

3.27

3.38

3.23

3.04

3.16

placenta weight

(mean) [g]

not recorded

 

 

 

 

 

crown-rump length (mean)[mm]

not recorded

 

 

 

 

 

Foetal sex ratio

[ratio m/f]

not reported

 

 

 

 

 

Examination of the foetuses:

Parameter

Control

0.025

dose

0.05

dose

0.075

dose

0.10

dose

0.20

dose

External malformations       mean

% offspring per litter           ±SEM

0.4

0.3

0.0

0.0

0.4

0.4

0.4

0.3

0.0

0.0

3.7

3.7

External variations               mean

% offspring per litter            ±SEM

0.2

0.2

0.0

0.0

0.0

0.0

0.5

0.3

0.0

0.0

0.0

0.0

Skeletal malformations         mean

% offspring per litter            ±SEM

2.0

0.7

0.9

0.6

1.6

0.6

2.5

0.7

3.5

1.2

4.3

1.5

Skeletal variations                mean

% offspring per litter            ±SEM

10.0

2.0

3.4

1.0

6.5

1.8

5.3

1.4

7.4

2.1

12.1

3.0

Visceral malformations         mean

% offspring per litter            ±SEM

39.9

7.0

40.6

6.3

44.3

6.5

42.9

6.6

45.4

6.9

46.4

6.5

Visceral variations               mean

% offspring per litter            ±SEM

1.4

0.8

2.1

1.2

2.9

1.5

0.0

0.0

1.3

0.7

0.5

0.5

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 % (55 mg/kg bw boric acid). At 0.100 % boric acid (76 mg/kg bw boric acid) there was reduced fetal bodyweight, short and wavy ribs, and these effects disappeared during the postnatal period. Similar but more marked effects were observed at the highest dose of 0.200 % (143 mg/kg bw boric acid) and apart from the short 13th rib, they also disappeared during the postnatal period.
Read-across is justified on the basis detailed in the rationale for reliability above. This study is therefore considered to be of sufficient adequacy and reliability to be used as a supporting study and no further testing is justified.