Diboron calcium tetraoxide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Numerous studies have demonstrated the toxicokinetics of boric acid and borate salts in humans and experimental animals. At physiological pHs in the aqueous layer of mucosal surfaces, prior to absorption, simple inorganic borated salts are hydrolysed to undissociated boric acid, which are readily absorbed via oral and inhalation exposure. Boric acid is not expected to hydrolyse/react further due to the high energy required to break the B – O bond. Therefore, it is expected that there will be negligible parent compound, calcium metaborate, available systemically and boric acid will be the dominant and common compound in humans and experimental animals. Therefore, a read-across approach using boric acid data is considered appropriate to use, refer to Section 13.

In a three-generation dietary study, groups of 8 males and 16 females were treated with the source substance, boric acid, at doses of 0, 34, 100 and 336 mg/kg bw/day (equivalent to 0, 5.9, 17.5 and 58.5 mg B/kg bw/day, respectively). Rats were exposed from the start of the study until sacrifice of parents P0 and from weaning until sacrifice for parents of F1 and F2 generations. F1a, F2a, F3a and F3b litters were sacrificed at weaning, while F1b and F2b litters were used for breeding. Parents of the high dose group were sterile; therefore, only controls, low and mid dose groups were taken to F2 and F3 generations. Testicular atrophy was reported in the high dose male P0 rats and microscopic examinations identified no viable sperm. In addition, there was decreased ovulation in females in the top dose group and only 1/16 females produced a litter when mated with control male rats. No adverse reproductive effects or gross malformations were reported in P0, F1 and F2 male and female rats in the low and mid-dose groups. High-dose male and female rats showed clinical signs of toxicity, including rough fur, scaly tails, respiratory distress and inflamed eyelids. High-dose parental rats were also sterile; therefore, only the controls, low and mid dose groups were taken to F2 and F3 generations. Based on the results a P0, F1 and F2 NOAEL of 100 mg/kg bw/day (equivalent to 17.5 mg B/kg bw/day) was identified.

Link to relevant study records

Reference

Reference 1

Endpoint:

three-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to justification attached in section 13.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

LOAEL

Effect level:

336 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 1170 ppm in the diet. Based on sterility.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

LOAEL

Effect level:

58.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: Based on sterility. Testicular atrophy, reduced fertility (no offspring from high dose females mated with untreated males).

Dose descriptor:

NOAEL

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

other: The authors reported no adverse effects on fertility, lactation, litter size, progeny weight or appearance in rats exposed to either 5.9 or 17.5 mg B/kg bw

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: No adverse effects in mid and low dose groups in any generation.

Reproductive effects observed:

not specified

Conclusions:

Rats exposed to the high dose of 336 mg/kg bw boric acid (corresponding to a level of 58.5 mg B/kg bw) were sterile. Microscopic examination of the atrophied testes of all males in this group showed no viable sperm. The authors also reported evidence of decreased ovulation in about half of the ovaries examined from the females exposed to 58.5 mg B/kg bw and only 1/16 matings produced a litter from these high dose females when mated with control male animals. There were no adverse effects on reproduction reported at exposures of 34 and 100 mg/kg bw boric acid (5.9 and 17.5 mg B/kg bw). The authors reported no adverse effects on fertility, lactation, litter size, progeny weight or appearance in rats exposed to either 5.9 or 17.5 mg B/kg bw. Also, no gross abnormalities were observed in the organs examined from either parents or weanlings from these dose groups. Based on these study data, the authors concluded that exposure of rats at levels up to 17.5 mg B/kg bw in the diet in a 3 generation reproduction study was without adverse effect.

Reference 2

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: No guideline specified, but conforms to the standard 3 generation 2 litters per generation multi-generation studies normally used at that time.

Deviations:

not applicable

GLP compliance:

no

Remarks:

Study pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Caesarean-derived from Charles River
Weight at study initiation: (P) Males: 121 - 150 g; Females: 110 - 147 g
- Diet: Ad libitum
- Housing: Prior to initiation of the first breeding phase, the animals were maintained in individual cages and fed their respective diets for 14 weeks until they reached maturity.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Rats were exposed from beginning of the study until sacrifice of parents P0 , and from weaning till sacrifice for the parents of the F1 and F2-generations.
The high dose group P animals were sterile so only controls, low and mid dose groups were taken to the F2 and F3 generations.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): The test material was incorporated into the basal diet on a weight/weight basis and thoroughly mixed in a twin-shell blender to provide the desired dietary levels.

Details on mating procedure:

- M/F ratio per cage: 1:2
- Length of cohabitation: 21 days on each occasion
- Any other deviations from standard protocol: This is a three generation multigeneration study with two matings (two litters) per generation. The F1a, F2a and F3a litters were sacrificed at weaning, and the F1b and F2b litters raised and used for breeding, and the F3b killed at weaning.

24 h after birth, the litters were reduced to a maximum of eight pups to be nursed. The F1A litters were discarded when they reached 21 days of age. The parents in the control and two lower test groups were remated to produce their second (F1B) litters. At the time of weaning 16 females and 8 males from the control and two test groups were selected at random and designated as the second parental generation (P2) for continuation of the reproduction study. All excess weanlings were discarded.
The experimental design for the high level test group (0.67 %) was altered due to failure of the P1 parents to produce litters. In order to determine whether the female reproductive system was affected, the P1 females in the high level group were mated with males of the same strain and approximately the same age, which had received only the control diet. The males remained in the breeding cage for 8 h each day. To prevent the males from feeding on teh test diet, no food was available to the animals during the daily mating period.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Groups of 8 males and 16 females were used for all generations and were exposed from beginning of the study until sacrifice of parents P0, and from weaning till sacrifice of the F1- and F2-generations.
The high dose group P animals were sterile so only controls, low and mid dose groups were taken to the F2 and F3 generations.

Frequency of treatment:

Daily

Details on study schedule:

This is a three generation multigeneration study with two matings (two litters) per generation. The F1a, F2a and F3a litters were sacrificed at weaning, and the F1b and F2b litters raised and used for breeding, and the F3b killed at weaning.
From beginning of the study until sacrifice of parents P0, and from weaning till sacrifice for the parents of the F1 and F2-generations.
The high dose group P animals were sterile so only controls, low and mid dose groups were taken to the F2 and F3 generations.

Remarks:

Doses / Concentrations:
0, 670, 2000 or 6700 ppm boric acid (0, 117, 350 and 1,170 ppm boron) in the diet, equivalent to 0, 34 (5.9), 100 (17.5) and 336 (58.5) mg boric acid (mg B)/kg bw/day.
Basis:

No. of animals per sex per dose:

8 males and 16 females per group

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment: By stratified randomisation

Positive control:

No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, weekly
- 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

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Sperm parameters were not done in the high dose group in which histology of the testes were performed.

Litter observations:

Number and sex of pups, stillbirths, live births, presence of gross abnormalities, weight gain, physical or behavioural abnormalities; culled to 8 per litter at 42 h after delivery.
Records were maintained on the number of conceptions, number and size of litters, deaths and weights of the pups at 24 h and at weaning. The pups were observed for gross signs of abnormalities.

Postmortem examinations (parental animals):

After completion of the second cycle (F1B) of the first breeding phase, all P1 animals in the control and two lower test groups were sacrificed (34th week of study). The males in the high level group were sacrificed after completion of the 27th week and the females after completion of the 46th week of the study. Gross necropsies were performed and representative tissues from each rat were preserved in 10 % formalin. Weights were obtained for brain, thyroid, liver, spleen, kidneys, adrenals and testes in all groups; and ovaries and uterus in the high level group. Organ/body weight ratios were obtained. Individual blood samples and pooled samples of brain, liver and kidney (all groups) and testis, ovary and uterus (high level only) were frozen for possible future analysis. The ovaries and uteri preserved from the high level females were examined microscopically.
After completion of the second breeding phase, all P2 animals were sacrificed and after completion of the third breeding phase, all P3 animals were savrificed. Necropsies were performed and the animals were observed for gross signs of pathology. The following tissues from eight males and eight females in the P2 and P3 control and test groups were preserved in 10 % formalin: Brain, thyroid, lung, heart, liver, kidney, adrenal, stomach, pancreas, small intestine, large intestine and gonad. Necropsies were also performed on 5 male and 5 female F3B weanlings from the control and two lower level test groups and representative tissues preserved in 10 % formalin.

Postmortem examinations (offspring):

No data

Statistics:

Terminal body weights, organ weights and organ/body weight ratios for the P1 animals were examined by the analysis of variance, of F-test, at the 5 % probability level. Before completing each F test, the variances were tested for heterogeneity by the method of Bartlett. If the variances were homogeous, the F-test could be applied in the normal fashion, and if a significant F value was obtained those groups significantly different from control could be determined by the method of Scheffe.
In those instances of heterogeneous variances, the samples were examined for extreme values by Sachs' test for rejection of measurements. If no legitimate unbiased adjustment to the variance could be made by rejection of "outliers", comparison test to control was effected by the Fisher-Behrens modified t-test. Breeding indices were analysed by the chi-square test of significance.

Reproductive indices:

No data

Offspring viability indices:

No data

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Parent males:
Rats of the P0 generation exposed to the high dose of 336 mg/kg bw boric acid (corresponding to a level of 58.5 mg B/kg bw) had reduced bodyweights though food intake was not affected and they were sterile. Microscopic examination of the atrophied testes of all males in this group showed no viable sperm. There were no adverse effects on reproduction reported at exposures of 5.9 and 17.5 mg B/kg bw. The authors reported no adverse effects on fertility, lactation, litter size, progeny weight or appearance in rats exposed to either 5.9 or 17.5 mg B/kg bw. Also, no gross abnormalities were observed in the organs from these dose groups.

Parent females:
The high dose groups of the P0 generation had reduced bodyweight without any effect on food intake. Evidence of decreased ovulation in about half of the ovaries examined from the females exposed to 58.5 mg B/kg bw and only one of 16 females produced a litter when mated with control male animals. There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

Dose descriptor:

LOAEL

Effect level:

336 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 1170 ppm in the diet. Based on sterility.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

LOAEL

Effect level:

58.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: Based on sterility. Testicular atrophy, reduced fertility (no offspring from high dose females mated with untreated males).

Dose descriptor:

NOAEL

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

other: The authors reported no adverse effects on fertility, lactation, litter size, progeny weight or appearance in rats exposed to either 5.9 or 17.5 mg B/kg bw

Clinical signs:

effects observed, treatment-related

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Description (incidence and severity):

There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

Histopathological findings:

not examined

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

F1 males:
There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.
F1 females:
There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

F2 males:
There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.
F2 females:
There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

The high dose group (58.5 mgB/kg bw) males and females showed clinical signs of toxicity with rough fur, scaly tails, respiratory distress and inflamed eyelids.
The high dose group P animals were sterile so only controls, low and mid dose groups were taken to the F2 and F3 generations.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: There were no adverse effects on reproduction and no gross abnormalities were observed in the organs at exposures of 5.9 and 17.5 mg B/kg bw.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: No adverse effects in mid and low dose groups in any generation.

Reproductive effects observed:

not specified

Table for reproductive toxicity:

Parameter			control		low dose		medium dose		High dose
		Generation	m	f	m	f	m	f	m	f
Mortality	incidence	P	0	1	0	0	0	0	0	0
		F1	0	1	0	0	0	0	0	0
		F2	0	0	0	0	0	0	0	0
Food consumption	% of control	not affected
Body weight gain	% of control		-	-	-	-	-	-	¯	¯
Clinical Observations specify effects	Incidence		-	-	-	-	-	-	+	+
Organ weights	% of control	only effect noted was increase in absolute wt. of thyroid in low dose group and relative thyroid wt. in low and mid dose groups (not thought to biologically significant)
Pathology
Histopathologic examination specify effects	Incidence	Evidence of testis atrophy in high dose males of P0 generation. Evidence in ovary of reduced ovulation in high dose females.
Reproductive Performance		P0 to F1a				F1b to F2b			F2b to F3b
		cont	low	mid	high	cont	low	mid	cont	low	mid
Mating index: (No. pregnant/No. mated)	%	62	88	81	0	80	94	94	69	94	94
Fertility index: No. litters born/No. Pregnant	%	100	100	100	-	100	100	100	91	100	100
Number of implantation sites	Mean
Duration of pregnancy	Mean
Birth index
Live birth index: No.pups alive/No. born	%	98	96	97		99	99	98	100	99	99
Gestation index
Litter size	Mean	12	11	11		12	13	12	12	13	11
Litter weight	Mean
Pup weight at 24h (g)	Mean	7.0	7.2	6.7		6.4	6.5	6.7	6.0	7.0	7.0
Sex ratio	Male/female	6/6	6/5	5/6		6/6	7/6	6/6	6/6	7/6	6/5
Survival index
Viability index
Lactation index: Pup wt. at weaning		55	50	52		56	53	51	48	51	55

Conclusions:

Rats exposed to the high dose of 336 mg/kg bw boric acid (corresponding to a level of 58.5 mg B/kg bw) were sterile. Microscopic examination of the atrophied testes of all males in this group showed no viable sperm. The authors also reported evidence of decreased ovulation in about half of the ovaries examined from the females exposed to 58.5 mg B/kg bw and only 1/16 matings produced a litter from these high dose females when mated with control male animals. There were no adverse effects on reproduction reported at exposures of 34 and 100 mg/kg bw boric acid (5.9 and 17.5 mg B/kg bw). The authors reported no adverse effects on fertility, lactation, litter size, progeny weight or appearance in rats exposed to either 5.9 or 17.5 mg B/kg bw. Also, no gross abnormalities were observed in the organs examined from either parents or weanlings from these dose groups. Based on these study data, the authors concluded that exposure of rats at levels up to 17.5 mg B/kg bw in the diet in a 3 generation reproduction study was without adverse effect.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rat

Quality of whole database:

Reliable with restrictions

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Numerous studies have demonstrated the toxicokinetics of boric acid and borate salts in humans and experimental animals. At physiological pHs in the aqueous layer of mucosal surfaces, prior to absorption, simple inorganic borated salts are hydrolysed to undissociated boric acid, which are readily absorbed via oral and inhalation exposure. Boric acid is not expected to hydrolyse/react further due to the high energy required to break the B – O bond. Therefore, it is expected that there will be negligible parent compound, calcium metaborate, available systemically and boric acid will be the dominant and common compound in humans and experimental animals. Therefore, a read-across approach using boric acid data is considered appropriate to use, refer to Section 13.

Two developmental studies, which were similarly designed and conducted in the same laboratory, were assessed using the benchmark dose (BMD) approach. As part of Study A 29 mated females/dose group were exposed to the source substance, boric acid, continuously in the diet at 0, 78, 163, 330 and 539 mg/kg bw/day. Study B included 60 mated females/dose group exposed to the source substance, boric acid, in the diet at 0, 19, 36, 55, 76 and 143 mg/kg bw/day. Both studies consisted on phase I developmental toxicity termination on gestation day 20 and phase II postnatal recovery termination on postnatal day 21. Effects reported in foetuses included enlarged lateral ventricles in the brain, shortening of rib XIII and variations of the first lumbar rib. Decreased foetal body weight was used for the basis for the BMD analysis and a BMDL05 of 59 mg/kg bw/day (equivalent to 10.3 mg B/kg bw/day) was identified.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to justification attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Dose descriptor:

BMD:

Effect level:

59 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Decreased foetal body weight provided the best basis for BMD calculations. The benchmark dose is defined as the 95% lower bound on the dose corresponding to a 5% decrease in the mean foetal weight (BMDL05).

Remarks:

Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Dose descriptor:

BMD:

Effect level:

10.3 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Dose descriptor:

BMD:

Effect level:

59 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Decreased foetal body weight provided the best basis for BMD calculations. The benchmark dose is defined as the 95% lower bound on the dose corresponding to a 5% decrease in the mean foetal weight (BMDL05).

Remarks:

Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Dose descriptor:

BMD:

Effect level:

10.3 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Developmental effects observed:

not specified

Conclusions:

Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the use of NOAELs divided by uncertainty factors. The benchmark dose approach has been proposed as an alternative basis for reference value calculations. In the analysis presented of the developmental toxicity of rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various endpounts (rib XIII effects, variations of the first lumbar rib) and foetal weight changes and various modelling approaches for those endpoints the best approach for incorporating all the information was determined. Decreased foetal body weight provided the best basis for BMD calculations. The BMD was calculated as 59 mg/kg bw/day.

Executive summary:

A benchmark dose developed by Allen et al. (1996) was based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996). The benchmark dose is defined as the 95 % lower bound on the dose corresponding to a 5 % decrease in the mean foetal weight (BMDL₀₅). The BMDL₀₅of 10.3 mg/kg body weight per day as boron is close to the Price et al. (1996) NOAEL of 9.6 mg/kg body weight per day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline required

Principles of method if other than guideline:

Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the use of NOAELs divided by uncertainty factors. The benchmark dose (BMD) approach has been proposed as an alternative basis for reference value calculations. In this analysis of the developmental toxicity observed in rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various endpoints (rib XIII effects, variations of the first lumbar rib) and foetal weight changes and various modelling approaches for those endpoints, the best approach for incorporating all of the information available from the studies was determined. In this case, the approach involved combining data from two studies which were similarly designed and were conducted in the same laboratory to calculate BMDs that were more accurate and more precise than from either study alone.

GLP compliance:

not specified

Remarks:

not applicable (it is a publication)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

20 days

Frequency of treatment:

continuously in diet

Remarks:

Doses / Concentrations:
0, 0.025, 0.05, 0.075, 0.1 and 0.2% equivalent to 0, 19, 36, 55, 76 and 143 mg Boric acid/kg bw (Price et al., 1994, 1995) - Study B
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 0.1, 0.2, 0.4 and 0.8 % equivalent to 0, 78, 163, 330 and 539 mg Boric acid/kg bw (Heindel et al., 1992) - Study A
Basis:
nominal in diet

No. of animals per sex per dose:

- 29 time-mated females/group (study A);
- 60 time-mated females/group (study B).

Control animals:

yes, plain diet

Details on study design:

The studies consist of two phases:
- Phase I: developmental toxicity termination on gd 20;
- Phase II: Postnatal recovery termination on pnd 21 (has not been considered in the analyses dicussed in the publication)

Dose descriptor:

BMD:

Effect level:

59 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Decreased foetal body weight provided the best basis for BMD calculations. The benchmark dose is defined as the 95% lower bound on the dose corresponding to a 5% decrease in the mean foetal weight (BMDL05).

Remarks:

Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Dose descriptor:

BMD:

Effect level:

10.3 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- incidence of total malformations, enlarged lateral ventricles in the brain, agenesis or shortening of rib XIII , and variations of the first lumbar rib, as well as decreased fetal weights.

Dose descriptor:

BMD:

Effect level:

59 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Decreased foetal body weight provided the best basis for BMD calculations. The benchmark dose is defined as the 95% lower bound on the dose corresponding to a 5% decrease in the mean foetal weight (BMDL05).

Remarks:

Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Dose descriptor:

BMD:

Effect level:

10.3 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996) (cited in Allen et al., 1996).

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference concentrations based on the ues of NOAELs divided by uncertainty factors. The benchmark dose approach has been proposed as an alternative basis for reference value calculations. In the analysis presented of the developmental toxicity of rats exposed to boric acid in their diet, BMD analyses have been conducted using two existing studies. By considering various endpounts (rib XIII effects, variations of the first lumbar rib) and fetal weight changes and various modelling approachesfor those endpoints the best approach for incorporating all the information was determined. Decreased foetal body weight provided the best basis for BMD calculations. The BMD was calculated as 59 mg/kg bw/day.

Executive summary:

A benchmark dose developed by Allen et al. (1996) was based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price et al. (1996). The benchmark dose is defined as the 95 % lower bound on the dose corresponding to a 5 % decrease in the mean fetal weight (BMDL₀₅). The BMDL₀₅of 10.3 mg/kg body weight per day as boron is close to the Price et al. (1996) NOAEL of 9.6 mg/kg body weight per day.

Reference 3

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to justification attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

other: Crl: CD VAF/Plus (Sprague Dawley)

Dose descriptor:

LOAEL

Effect level:

143 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity - Based on relative kidney weights.

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:

25 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity - Based on relative kidney weights.

Dose descriptor:

NOAEL

Effect level:

13.3 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity

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

Dose descriptor:

LOAEL

Effect level:

76 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental effects

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:

13.3 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity - Based on reduced foetal body weight and increased incidence of short rib XIII.

Dose descriptor:

NOAEL

Effect level:

9.6 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

Developmental effects observed:

not specified

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 foetal 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.

Reference 4

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: CD VAF/Plus (Sprague Dawley)

Details on test animals or test system 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

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 foetal 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

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 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 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.

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

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).

Dose descriptor:

LOAEL

Effect level:

143 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity - Based on relative kidney weights.

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 - Based on relative kidney weights.

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 - Based on reduced foetal body weight and increased incidence of short rib XIII.

Dose descriptor:

NOAEL

Effect level:

9.6 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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.

Reference 5

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to justification attached in section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rabbit

Strain:

New Zealand White

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

21.8 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

>= 21.8 mg/kg bw/day (nominal)

Based on:

element

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

>= 125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Developmental effects observed:

not specified

Conclusions:

The highest dose was very toxic to dams and 90 % of implants were resorbed at the highest dose level, and 72 % of surviving foetuses had cardiac or great vessel malformations or increase in resorptions were reported in the mid and low dose groups.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products Inc., Denver, PA, USA
- Age at study initiation: 5 months of age
- Weight at study initiation: 2690-4380 g

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Concentration in vehicle: 55 mg/mL boric acid
- Amount of vehicle: 5 mg/mL boric acid

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Details on mating procedure:

- Impregnation procedure: Artificial insemination; designated Day 0

Duration of treatment / exposure:

Groups of 30 rabbits were used treated on Day 6 - 19 post-mating

Frequency of treatment:

No data

Duration of test:

Terminated on Day 30 of gestation

Remarks:

Doses / Concentrations:
0, 62.5, 125 or 250 mg/kg bw boric acid equivalent to 0, 10.9, 21.8 and 43.5 mg B/kg bw
Basis:
no data

No. of animals per sex per dose:

30 females/group

Control animals:

yes

Details on study design:

No data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 30
- Organs examined: 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 early resorptions: No data
- Number of late resorptions: No data
- Other:
Uteri were stained with ammonium sulphide as no implantations were visible.
Litter size, number of dead foetuses and foetal weight were assessed.

Fetal examinations:

- External examinations: Yes, including assessment for cleft palate
- Soft tissue examinations: Yes by dissection (Staples) and sex determined
- Skeletal examinations: Yes, all foetuses were skinned and cleaned and stained with alcian blue/alizarin red S
- Head examinations: Yes

Statistics:

No data

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnant does exhibited no overt symptoms attributable to boric acid toxicity except in the high dose group. A decreased food intake (30 % reduction vs. controls during exposure period) and decreased maternal bodyweight were observed. Vaginal bleeding was noted at 43.5 mg B/kg bw between gestational days 19 - 30. All high-dose animals with vaginal bleeding had no live foetuses at sacrifice. At mid dose, increased body weight gain not clearly adverse. The authors considered 43.5 mg B/kg bw as the LOAEL for pregnant does and 21.8 mg B/kg bw as the NOAEL for maternal toxicity.

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

21.8 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Dose descriptor:

NOAEL

Effect level:

21.8 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At the highest dose in this study of 250 mg/kg bw boric acid (43.5 mg B/kg bw/day), 90 % of implants/litter were resorbed compared to 6 % for controls, and 73 % had complete litter loss (0 % in controls). In the mid and low dose groups, no difference in percentage resorptions per litter was seen, compared to controls.
Average foetal bodyweight per litter was 92 % of the controls at the high dose (43.5-mg B/kg bw) but even at this exposure, it did not reach statistical significance possibly due to the low number of pups surviving (14 fetuses from 6 litters).
An increased incidence of malformed live foetuses/litter was observed at 43.5 mg B/kg bw, primarily due to cardiovascular defects (72 % for major defects of heart and/or great vessel in the high-dose group vs. 3 % in controls). In the mid and low dose groups, there was no increase in malformations per litter or total malformations. There were no variations between any groups concerning the incidence of skeletal malformations.
The only skeletal variations of interest was a dose related reduction in the incidence of extra ribs on Lumbar I which the authors did not consider to be toxicologically important.
Since no definitive developmental effects were observed in animals exposed to either 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), the authors concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

>= 21.8 mg/kg bw/day (nominal)

Based on:

element

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

>= 125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Abnormalities:

not specified

Developmental effects observed:

not specified

Maternal effects

Parameter	Study control data	Low dose	Medium dose	High dose
Number of dams examined	30	30	30	30
Clinical findings during application of test substance				reduced food and bodyweight
Mortality of dams (%)	0	1	1	0
Abortions	0	0	0	3
Body weight gain               day 6-19                                              day 0-30	93 357	132 493	97 543	-137* 226
Food consumption g/kg/day  day 0-6                                                  day 6-19                                                  day 19-25	48.1 38.8 36.9	48.0 40.0 37.0	48.9 38.7 40.0	46.4 26.6* 44.9
Pregnancies  % pregnant at sacrifice	75	89	87	96
Necropsy findings in dams dead before end of test		gavage error lungs	stomach damage

* P < 0.05.

 

Litter response (Caesarean section data)

Parameter	Control data	Low dose	Medium dose	High dose
Corpora lutea                        number                                                  ±SEM mean No. per dam	12.2 0.7	10.7 0.5	11.5 0.4	10.0* 0.7
Implantations sites per litter     number                                                      ±SEM total/number of dams	9.5 0.8	8.4 0.6	8.3 0.5	8.6 0.7
Resorptions % per litter               % total/number of dams          ±SEM	6.3 2.4	5.9 1.9	7.7 2.1	89.9 5.0
total number of foetuses	159	175	153	14
total number of litters	18	23	20	6
live foetuses / litter                 number state ratio                               ±SEM	8.8 0.8	7.6 0.6	7.7 0.5	2.3* 0.8
dead foetuses / litter        % state ratio	0	2.8	0.4	0
foetus weight (mean)                   weight (g)                                                         ±SEM	44.8 1.5	46.5 1.4	45.7 1.2	41.1 2.7
Foetal sex ratio    % male per litter                                    ±SEM	50 5	51 4	55 4	69 10

* P<0.05

 

Examination of the foetuses

Parameter	Control data	Low dose	Medium dose	High dose
External malformations^* % foetuses per litter  ±SEM	0.8 0.8	1.4 1.0	1.0 1.0	11.1* 8.2
External malformations    No. of foetuses	1	2	1	2
Skeletal malformations^* % foetuses per litter ±SEM	19.9 5.4	19.9 4.0	24.3 6.4	38.9 20.0
Skeletal malformations     No. of foetuses	30	39	44	4
Visceral malformations^* % foetuses per litter  ±SEM	7.3 1.9	5.9 2.0	7.4 2.0	80.6* 16.3
Visceral malformations      No. of foetuses	13	11	12	11
% foetuses with cardiovascular       % malformations                               ±SEM	2.7 1.6	3.1 1.5	4.2 1.3	72.2* 16.5

* P<0.05

Conclusions:

The highest dose was very toxic to dams and 90 % of implants were resorbed at the highest dose level, and 72 % of surviving foetuses had cardiac or great vessel malformations or increase in resorptions were reported in the mid and low dose groups.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL05

59 mg/kg bw/day

Species:

rat

Quality of whole database:

Reliable with restrictions

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study (OECD test guideline 414), female rats (28-32/dose for both phase I and II) were exposed via the diet to the source substance, boric acid, at doses of 0, 19, 36, 55, 76 and 143 mg/kg bw/day (equivalent to 0, 3.3, 6.3, 9.6, 13.3 and 25 mg B/kg bw/day, respectively) during days 0 to 20 post mating (phase I) and days 0 to 20 post mating, then on a normal diet until day 21 postpartum (phase II). As part of phase I, dams were sacrificed on day 20 for foetal examination, phase II dams delivered pups and were reared to weaning until they were sacrificed to assess visceral and skeletal effects. In dams an increase in relative kidney weight was observed in phase I of the high dose group, but these effects were not observed in phase II. No additional maternal effects were reported. In phase I decreased foetal body weight per litter and skeletal effects (increased incidences of short rib XIII and wavy rib) were identified in the top two dose groups. In phase II an increased incidence of short rib XIII was reported at the top dose group. Based on these effects a maternal NOAEL of 76 mg/kg bw/day (equivalent to 13.3 mg B/kg bw/day) and a developmental NOAEL of 55 mg/kg bw/day (equivalent to 9.6 mg B/kg bw/day) were identified.

In another prenatal developmental toxicity study (OECD test guideline 414) female New Zealand White rabbits (30/dose) were exposed via oral gavage to the source substance, boric acid, at doses of 0, 62.5, 125 and 250 mg/kg bw/day (equivalent to 0, 10.9, 21.8, 43.5 mg B/kg bw/day, respectively) during days 6 to 19 post mating and were terminated on gestation day 30. In the high dose group decreased maternal body weight and food intake were reported. Vaginal bleeding was observed in the top dose between gestation days 19 to 30 and no live foetuses at termination. At the top dose 90 % of implants per litter were resorbed and 73 % had complete litter loss. Increased incidences of cardiovascular defects were also identified in the high dose group. No adverse effects were reported in foetuses in the low and mid dose groups. Based on these effects a maternal NOAEL of 43.5 mg/kg bw/day (equivalent to 21.8 mg B/kg bw/day) and a developmental NOAEL of 125 mg/kg bw/day (equivalent to 21.8 mg B/kg bw/day) were identified.

Justification for classification or non-classification

In accordance to Regulation (EC) No 1272/2008, the source substance, boric acid, is classified as Repr. 1B H360FD with a current Specific Concentration Limit of 5.5 %, under the 1st ATP to CLP. Boric acid is considered a suitable analogue; therefore, it is concluded that the target substance is aligned as Repr. 1B H360FD classification and a current SCL of 5.5 %.