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Effects on fertility

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Reference
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:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions. 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. 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.
Qualifier:
according to
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 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
Gross pathological findings:
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
Histopathological findings:
not examined
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.
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.
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
56.3 mg/kg bw/day
Species:
rat
Quality of whole database:
The most reliable comparable to guideline study with acceptable restrictions.
Additional information

Effects on male fertility have been investigated in detail. A dose related effect on the testis was observed in rats, mice and deer mice, with confirmation from limited studies in dogs. Effects in rats start with reversible inhibition of spermiation after 14 days (at 39 mg B/kg bw/day) and 28 days (at 26 mg B/kg bw/day). At doses equal to and above 26 mg B/kg bw/day testicular atrophy, degeneration of seminiferous tubules and reduced sperm counts were observed. Male fertility was further investigated in two serial mating studies of treated male rats with untreated female rats. Infertility of treated males correlated well with germinal aplasia. Similar effects on male fertility were described in deer mice (Peromyscus maniculatus) after treatment with boric acid. Fertility studies in rats (two three-generation study with for boric acid and disodium tetraborate decahydrate) and mice (a continuous breeding study with boric acid) further support effects on testes as the underlying cause for reduced male fertility.

Diminished sperm production may be due to testicular effects on germ cell, Sertoli cell, or Leydig cell function or act via an alteration of the pituitary-hypothalamic axis. There is an indication that LH and FSH are elevated under boric acid treatment (Lee et al., 1978) and that serum testosterone may be decreased in CD-1 mice and F344 rats (Grizzle et al., 1989; reviewed in Fail et al., 1991; Treinen & Chapin, 1991). The decrease in prostate weight at 111.3 mg B/kg bw/day observed by Fail et al. (1991) might be caused by reduced testosterone levels.

 

A NOAEL of 17.5 mg B/kg bw/day for effects on female fertility was derived in the Transitional Annex XV dossier (TD 2008) based on Weir (1966c-d) and Fail et al,1991.   However, the TD failed to adequately distinguish between effects on female fertility and effects on development. Fertility is generally defined in males as the ability to produce sperm which are capable of producing fertilisation of an ovum leading to conception.  In females, it is defined as the ability to produce and release ova which can be fertilised leading to conception.  To test fertility in animals males and females are pretreated to cover the period of development of the sperm and eggs, then mate and treat until the time of implantation, around Day 6 following mating, and then stop treatment in the females.   To test for effects on development pregnant females are treated from Day 6 till the end of pregnancy. Neither the Weir and Fisher multigeneration study nor the Fail RACB studies were performed with this division of treatments.  They both treated animals continuously before and during pregnancy and also after delivery.

In a three generation study in rats groups of 8 males and 16 females were treated with boric acid or disodium tetraborate decahydrate equivalent to 0, 5.9, 17.5 and 58.8 mg B/kg bw/day (Weir 1966c,d). An attempt was made to study the fertility of the P1 females at the top dose level by mating them with untreated males but only one litter of 16 pairs was produced. This highest dose level was clearly clinically toxic to the females after 2-3 weeks of dosing, with rough fur, scaly tails, inflamed eyelids and staining of the fur on the face and abdomen. The mating procedure to test the fertility of the females was not a satisfactory one. To avoid treatment of the males used for pairing, food was withdrawn from the cages of the females for 8 hours per day during the pairing process, and this is known to be very stressful to laboratory rats. There was no evidence on whether mating actually occurred for any of the rats, and no vaginal examinations for the presence of sperm were carried out. The females of the top dose P1 generation were sacrificed after 45 weeks of treatment and histopathological examination of the ovaries and uterus carried out. In the ovaries the presence of corpora lutea was regarded as a major indication of cyclic function, and these were found in 7 of 15 females, with reduced or absent function in the remaining 8 animals. The changes in the ovaries were not clearly different from those of controls.  No treatment related changes were found in the uterus. No changes were found that could account for the reduced litter production, and no conclusions could be drawn about fertility in the top dose females.  Comparable results were found in the Weir and Fisher multigeneration study on borax, with clear testicular atrophy at the top dose levels in males, and no clear explanation of the reduced number of litters in the top dose females, using the same unsatisfactory mating technique.  The authors of the study concluded that testis atrophy was clearly produced in males at the top dose level, but that the evidence of the decreased ovulation in females did not account for the reduced number of litters in the cross mating study in females.  Thus the Weir and Fisher studies produced clear evidence of adverse effects on male fertility, but did not produce clear evidence for an adverse effect on female fertility.

 

In a continuous breeding study of boric acid in Swiss mice (NTP, 1990; Fail et al., 1991), the three administered doses were 1000 ppm (26,6 mg B/kg bw/day), 4500 ppm (111,3 mg B/kg bw/day) and 9000 ppm (220,9 mg B/kg bw/day). A dose-related effect on the testis (testicular atrophy and effects on sperm motility, morphology and concentration) was noted; fertility was partially reduced at 111 mg B/kg bw/day, and absent at 221 mg B/kg bw/day.

 

For cross over mating only the mid dose group (111,3 mg B/kg bw/day) could be mated with control animals, since the high dose produced no litter. Indices of fertility for mid dose males with control females, control males with mid dose females and control males with control females were 5%, 65% and 74%, respectively. The according indices of mating (incidence of copulatory plugs) were 30%, 70% and 79%. This indicates that the primary effect was seen in males, however, slight effects were also noted in females. Live pup weight (adjusted for litter size) was significantly reduced compared to control litters, the average dam weight was significantly lower on postnatal day 0 compared to control dams and the average gestational period of the mid dose females was 1 day longer than in control females. The latter finding has also been observed in the developmental toxicity study by Price et al. (1996, see section 5.9.2).

 

In task 4 of this continuous breeding study control animals and low-dose F1 animals were mated because in the 9000 ppm groups no litters and in the 4500 ppm group only 3 litters were produced. While mating, fertility and reproductive competence were un-altered compared to control, the adjusted pup-weight (F2) was slightly but significantly decreased. F1 females had significantly increased kidney/adrenal and uterus weights and the oestrus cycle was significantly shorter compared to control females. A crossover mating study of controls and 4500 ppm groups confirmed the males as the affected sex.Necropsy at 27 weeks confirmed reduced testes weight, seminiferous tubule degeneration, decreased sperm count and motility and increase in abnormal sperm.In females at 27 weeks, 4500 ppm boric acid was toxic with decreased liver, kidney and adrenal weights, but no effect on oestrous cycles, mating, number of litters and number of pups. In F1 males a reduction in sperm concentration was observed, but no other sperm parameters were influenced.

 

While in this study the NOAEL for females of the F0-generation is 1000 ppm this is a LOAEL for males of the F0-generation (motility of epididymal sperms was significantly reduced: 78% ± 3 in controls vs. 69% ± 5 at 1000 ppm). For the F1-generation 1000ppm can be identified as a LOAEL, based on the 25% reduction of sperm concentration in males at this dose. Further, though normal in number, the F2-pups had reduced adjusted bodyweights at 1000 ppm, which is therefore also a LOAEL for F2-generation.

The authors concluded that the male is the most sensitive sex and that the testis is the primary target organ for boron. The NOAEL for testicular pathology in the present mouse study is probably 1000 ppm (26mg B/kg bodyweight). While males are more sensitive to boron induced toxicity, data also suggest an effect of boron on the female reproductive system. A reduced number of pups per litter and number of pups born alive at high dose levels are in agreement with earlier reports and could result from an effect of boron to alter implantation or to disrupt cell division in the embryo. This is supported by results of developmental toxicity studies in rats and mice in which higher dose levels can reduce the number of implants. Although F1 females had significantly increased kidney/adrenal and uterus weights and the oestrus cycle was significantly shorter compared to control female, similar effects were not observed in the 4500 ppm dose group, therefore the NOAEL for fertility in females was the dose level in diet of 4500 ppm, 846 mg/kg bw of boric acid or equivalent to 148 mg B/kg bodyweight.

In conclusion, the effects described in the Fail study on fertility show that 4500 ppm (111.3 mgB/kg bw) is a NOAEL for the females, and that other small effects in females are the result of developmental toxicity for which a NOAEL of <1000ppm (26.6mg B/kg bw) may be valid.

No further studies on the effects of boron on female fertility were reported by the National Toxicology Program team who published several other studies on the mechanism of action of boron on male fertility and on spermatogenesis. No effects on steroidogenic function were found in Leydig cells, and no clear mechanism of action to cause testis atrophy was identified by Ku and Chapin (1994).

 

Protective Effects of Zinc against Boric Acid Related Effects on Fertility

An important difference between laboratory animals and humans is the intrinsically higher zinc concentration in humans compared to laboratory animals. Normal levels of zinc in soft tissues in humans are over two times greater than in comparative tissues in laboratory animals (Figure 2, also see Appendix G, Figure 11) (King et al. 2000; Ranjan et al. 2011; Yamaguchi et al. 1996; Florianczyk 2000). The high zinc concentrations in humans compared to laboratory animals is also found in the target organs of boric acid, including fetal tissue, epididymis, and testes (Ahokas et al. 1980; Dorea et al. 1987; Suescun et al. 1981). The protective effect of the intrinsically large zinc stores in the human body against boric acid associated toxicity explains in part the absence of effects in humans exposed to high levels of boron.

Boron industry worker studies show that boron appears to concentrate in the semen, however without corresponding adverse effects on the semen. This is likely due to the fact that zinc concentrations are higher in semen in humans (112 µg zinc / ml), protective against the adverse effects of boric acid (Sorensen et al. 1999).

To investigate the effect of zinc on boric acid related toxicity on fertility effects an in vitro spermatogenesis study with boric acid in the presence of varying amounts of zinc was conducted (Durand 2013).Seminiferous tubules were cultured in the presence of 32 µg/ml boric acid and varying concentrations of zinc chloride equivalent to 0.48, 1.2, 2.4 and 4.8 µg Zn / ml to determine the effects on the number of somatic cells (sertoli and myoid cells) and germ cells (pre-meiotic cells (spermatogonia), meiotic cells (young spermatocytes, middle to late pachytene spermatocytes and secondary spermatocytes), and post-meiotic cells (round spermatids)) after 1 and 2 weeks of culture. An absence of boric acid related effects on spermatogenesis was observed in the presence of zinc (Figure 3, see also Appendix G, Figure 12). All germ cells populations were decreased by boric acid at 32 μg / ml. At D14, in the presence of boric acid increasing concentrations of zinc chloride lead to:- A dose dependent increase in the number of germ cells, an increased number of spermatogonia, a dose dependent increase in the number of young spermatocytes, a dose dependent increase of the number of middle to late pachytene spermatocytes, a dose dependent increase of secondary spermatocytes, and a dose dependent increase of round spermatids. Details of this study are presented in Appendix G. These results suggest that zinc interacts with boric acid reducing boric acid induced toxicity on spermatogenesis.

Zinc Borate Toxicity Studies

In addition to the in vitro study , a 28-day oral (gavage) dose range finding toxicity study of zinc borate and 90-day toxicity study of zinc borate in Sprague-Dawley Rats was also recently completed (See Kirkpatrick 2013 in Chemical Safety Report - Zinc Borate). This study is presented in more detail in Appendix G. Dosage levels tested in the 28-day study were 125, 250, 500, 1000 mg ZB/kg bw equivalent to 18.65, 37.3, 74.6 and 149.2 mg Boron/kg bw. A NOAEL of 37.3 mg B/kg bw and LOAEL of 74.6 mg B/kg bw for male fertility effects determined by minimal histopathologic findings in testes and epididymides was observed. Since hypospermia was not observed at the LOAEL, and the histopathological changes were graded as minimal, the effects observed in the 75 mg B/kg bw group were not considered toxicologically significant (Kirkpatrick 2013). The male fertility NOAEL for boric acid is 17.5 mg B/kg bw. No microscopic findings were noted in the ovaries.

Dosage levels tested in the 90-day study were 50, 100, 200, and 375 mg ZB/kg bw equivalent to 7.46, 14.92, 29.84 and 55.95 mg Boron/kg bw. The objectives of this study were to evaluate the potential toxicity of zinc borate when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days and to assess recovery from such effects. 

Adverse test substance-related microscopic findings were noted in the 375 mg/kg/day group males and consisted of germ cell degeneration in the testes, decreased size of the epididymides, inflammation of the prostate, and debris in the prostate. Test substance-related effects on spermatogenic parameters were noted in the 200 and 375 mg/kg/day group males at the study week 13 necropsies, as indicated by lower percentages of motility, progressive motility, and normal sperm at 200 and 375 mg/kg/day and a lower sperm production rate at 375 mg/kg/day. No microscopic findings were found in the testes or epididymis in the 200 mg/kg/day dose group. These effects for the 375 mg/kg/day group were considered adverse due to correlating microscopic findings.

Based on the results of this study, oral administration of zinc borate to Sprague Dawley rats for a minimum of 90 consecutive days resulted in no adverse effects for females at dosage levels of 50, 100, 200, and 375 mg/kg/day. For males, a dosage level of 375 mg/kg/day resulted in adverse effects on male reproductive organs, including effects on spermatogenic parameters with corresponding lower organ weights and gross and microscopic findings. Adverse effects on spermatogenic parameters were also noted at 200 mg/kg/day although there were no correlating microscopic findings. Therefore, no-observed-adverse-effect level (NOAEL) was 100 mg/kg/day for males and 375 mg/kg/day for females. Absence of microscopic findings in the testes and epididymides of the 200 mg/kg/day dose group suggest that zinc interacts with boric acid reducing boric acid induced toxicity on male reproductive organs.

Of note, even with high exposures to zinc, tissue concentrations in the testes, epididymis, and ovaries of rats remain well below normal zinc levels found in comparative human tissues. Although zinc tissue concentrations were not measured in the 28-day or 90-day zinc borate studies, zinc levels were measured in select tissues in a 90 day inhalation toxicity study of zinc oxide (ZnO) in rats sponsored by the U.S. Library of Congress and conducted by Battelle Laboratories(Placke 1990). The purpose of the study was to evaluate the potential toxicity of inhaled zinc oxide aerosol following sub-chronic exposures in rats (5 days per week for 13 consecutive weeks), to define the concentration response, to identify and characterize effects on target organs, to determine the tissue distribution of zinc as a function of concentration and continued exposure, to determine the reversibility of exposure-related toxic effects, to evaluate specific toxic potential on the immune, hematopoietic, and reproductive systems and to select concentrations for a possible subsequent chronic toxicity and carcinogenicity study in Fischer 344 Rats. Animals were exposed for 6 hours/day, 5 days/week for 13 weeks, to ZnO aerosol at 0, 1, 3, 10, 50 and 200 mg/m3. Five rats/sex/group were measured to determine absorption, distribution, accumulation, excretion and clearance of zinc. Exposure to 200 mg/m3 of ZnO resulted in a significantly increased total body burden of zinc. Tissue levels of zinc increased in most tissues (with the exception of testes, epididymis, ovaries, and RBC) during exposure and returned to near control levels during the recovery period (with the exception of lung and bone which showed zinc retention). Tissues with the greatest increases were lung, liver, pancreas and femer. Even after 90-days of exposure at the highest inhalation exposure concentration of ZnO, no increase in zinc levels were seen in the testes, epididymis, ovaries, and RBC and still well below the intrinsic levels observed in humans (See graph Appendix G). A greater degree of protection from boric acid related fertility effects would be expected in human tissues with substantially greater concentrations of zinc.

Although boron has been shown to adversely affect male reproduction in laboratory animals, male reproductive effects attributable to boron have not been demonstrated in studies of highly exposed workers.


Short description of key information:
A multigeneration study in the rat (Weir, 1966) gave a NOAEL for fertility in males of 17.5 mg B/kg/day.

Justification for selection of Effect on fertility via oral route:
Three-generation study with the lowest NOAEL was chosen.

Effects on developmental toxicity

Description of key information
A benchmark dose of 33.1 mg/kg bw/day (10.3 mg B/kg bw/day) for developmental toxicity 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).
Link to relevant study records
Reference
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:
other: see 'Remark'
Remarks:
Meets acceptable scientific standards with acceptable restrictions. 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. 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.
Qualifier:
according to
Guideline:
other: No data
Deviations:
not specified
Principles of method if other than guideline:
Developmental toxicity risk assessment has typically relied on the estimation of reference doses or reference conncetrations based on the ues of NOAELs divided by uncertainty factors. The benchmark dose approach has been proposed as an alternative basis for refrence alue 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 approaches for those endpoints the best approach for incorporating all the information was determined.
GLP compliance:
not specified
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. 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 and modelling approaches for those endpoints, the best approach for incorporating all of the information available from the studies could be 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
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
No data
Basis:
no data
Control animals:
not specified
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 fetal weight (BMDL05).
Remarks:
Results are based on the studies of Heindel et al. (1992), Price, Marr & Myers (1994) and Price 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 fetal 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.
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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMDL05
33.1 mg/kg bw/day
Species:
rat
Quality of whole database:
The study meets acceptable scientific standards with acceptable restrictions.
Additional information

Developmental effects have been observed in three species, rats, mice and rabbits. The most sensitive species being the rat with a NOAEL of 9.6 mg B/kg bw/day. This is based on a reduction in mean foetal body weight/litter, increase in wavy ribs and an increased incidence in short rib XIII at 13.3 mg B/kg bw/day. The reduction in foetal body weight and skeletal malformations had reversed, with the exception of short rib XIII, by 21 days postnatal. At maternally toxic doses, visceral malformations observed included enlarged lateral ventricles and cardiovascular effects.

The NOAEL for this endpoint is 9.6 mg B/kg bw/day corresponding to 55 mg boric acid/kg bw/day, 31 mg boric oxide/kg bw/day, 85 mg disodium tetraborate decahydrate/kg bw/day, 65 mg disodium tetraborate pentahydrate/kg bw/day and 44.7 mg disodium tetraborate anhydrous/kg bw/day.

The critical effect is considered to be decreased fetal body weight in rats, for which the NOAEL was 9.6 mg/kg body weight per day. 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.

There is no evidence of developmental effects in humans attributable to boron in studies of populations with high exposures to boron(Tuccar et al 1998; Col et al. 2000; Chang et al. 2006).

Protective Effects of Zinc against Boric Acid Related Developmental Effects

Comparative zinc concentrations in humans and rat indicate that the protective effects of zinc are present early in the developing human fetus. Significantly higher concentrations (3x) of zinc in the human fetus compared to laboratory animals have been reported (Figure 2, see also Appendix G, Figure 11).

To investigate the effect of zinc on boric acid related toxicity on developmental effects, a GLP-compliant embryonic stem cell test (EST) conducted according to INVITTOX Protocol No. 113 was conducted to investigate the embryotoxic potential of boric acid in the presence of varying concentrations of zinc in vitro. No greater sensitivity of embryonic stem cells compared to fully differentiated cells was observed and no concern for in vivo embryotoxicity is triggered for boric acid at various concentrations of zinc. A reduction in the boric acid inhibition of differentiation of D3 embryonic stem cells was observed with increasing concentrations of zinc (Figure 4, see also Appendix G, Figure 13) (Hofman-Huther 2013).

The potential of zinc borate for developmental toxicity was evaluated based on the results of the Developmental Toxicity Test (Edwards 2014). Three treatment groups of 25 Sprague Dawley rats were administered the test article via oral gavage at dose levels of 100, 125 or 150 mg/kg/day. No test substance-related visceral developmental variations were noted in the 100, 125, and 150 mg/kg/day groups. There were no test substance-related skeletal malformations noted for foetuses in the test substance-treated groups. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Higher mean litter proportions of reduced ossification of the 13th rib(s) and sternebra(e) nos. 5 and/or 6 unossified were noted in the 100, 125, and 150 mg/kg/day groups compared to the control group. These findings were considered secondary to the reduced foetal weights noted in these groups. In addition, higher mean litter proportions of 7th cervical ribs and lower mean litter proportions of 14th rudimentary rib(s) were noted in the 100, 125, and 150 mg/kg/day groups and higher mean litter proportions of 25 presacral vertebrae were noted in the 125 and 150 mg/kg/day groups compared to the control group. No test substance-related foetal malformations were observed in the test substance-treated groups. Based on these results, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dosage level of <100 mg/kg/day was considered to be the NOAEL for embryo/foetal development when zinc borate was administered orally by gavage to inbred Crl: CD(SD) rats.


Justification for selection of Effect on developmental toxicity: via oral route:
BMDL05 established for boric acid is more accurate than the NOAELs from different developmental toxicity studies.

Justification for classification or non-classification

Boric oxide is classified under the 1stATP to CLP as Repr. 1B; H360FD.

While boron has been shown to adversely affect male reproduction in laboratory animals, there is no clear evidence of male reproductive effects attributable to boron in studies of highly exposed workers (Whorton et al. 1994; Sayli 1998, 2001; Robbins et al. 2010; Scialli et al. 2010, Duydu 2011). There is also no evidence of developmental effects in humans attributable to boron in studies of populations with high exposures to boron (Tuccar et al 1998; Col et al. 2000; Chang et al. 2006).

Comparison of Blood, Semen and Testes Boron Levels in Human and Rat

A comparison of blood, semen and target organ boron levels in studies of laboratory animals and human studies shows that boron industry worker exposures are lower than untreated control rats. Background boron levels in standard rat chow are high (10-20 ppm), as a result control rats in toxicity studies receive 45 times more boron than background exposure in humans. Blood boron levels in female control rats is about 0.23 µg B/g (Price et al. 1997), approximately equal to the blood levels in boron industry workers in China, Turkey and U.S. of 0.25, 0.22 and 0.26 µg B/g, respectively (Scialli et al. 2010; Culver et al. 1994; Duydu et al. 2011). Plasma and seminal vesicle fluid (the major component of semen) boron levels in untreated male control rats were 1.94 and 2.05 µg B/g, respectively, while boron levels in testes in rats dosed at the rat fertility LOAEL (26 mg B/kg) was 5.6 µg B/g (Ku et al. 1991,1993). Values in male control rats were higher than corresponding boron levels in the highest exposed Chinese boron industry workers with blood boron levels of 1.56 µg B/g and 1.84 µg B/g in semen (Scialli et al. 2010). Blood and semen boron levels in highly exposed Turkish boron workers were also lower than control rats with levels of 0.22 and 1.88 µg B/g, respectively (Duydu et al. 2011). Boron levels in testes of rats dosed at the rat fertility LOAEL was over 3x the blood boron levels in highest exposure group of Chinese boron industry workers. The blood level at the lowest animal LOAEL (13 mg B/kg) was 1.53 µg B/g, about 6 times greater than typical boron industry workers (Price et al. 1997). No adverse effects on sperm were seen in Turkish boron industry workers or in the most highly exposed subgroup of Chinese boron industry workers drinking boron contaminated water (mean blood level 1.52 µg B/g, the human NOAEL). Only under extreme conditions do human levels reach those of this animal LOAEL: the subgroup of Chinese boron workers who also drank contaminated water. Since no boron accumulation occurs in soft tissues (testes) over plasma levels biological monitoring in humans provide direct comparison to test animal target organ boron levels. Workers in boron mining and processing industries represent the maximum possible human exposure however their blood and semen boron levels are less than levels in untreated control rats. This provides an explanation why studies of highly exposed boron industry workers have shown no adverse effects and demonstrates that maximal possible exposures in humans are insufficient to cause reproductive toxicity effects. Graphs comparing the rodent and human exposure, blood, semen and tissue boron levels are presented in Appendix C. The human exposure data do not support classification of boric acid or boric oxide as Category 1(B) reproductive toxicant.

Extensive evaluations of sperm parameters in highly exposed workers have demonstrated no effects on male fertility. While no developmental effects have been seen in highly exposed populations, epidemiological studies of developmental effects are not as robust as the fertility studies. Reproductive effects data for the developmental epidemiological studies were obtained by self-reported data collected by personal interviews of workers and questionnaires, small sample sizes, and lack of actual exposure measurements during pregnancy limit the conclusions that can be made from the developmental studies in humans.

Protective Effects of Zinc against Boric Acid Related Developmental Effects

Comparative zinc concentrations in humans and rat indicate that the protective effects of zinc are present early in the developing human fetus. Significantly higher concentrations (3x) of zinc in the human fetus compared to laboratory animals have been reported (Figure 2, see also Appendix G, Figure 11).

Embryonic Stem Cell Test with Zinc Chloride and Boric Acid

A GLP-compliant embryonic stem cell test (EST) conducted according to INVITTOX Protocol No. 113 to investigate the embryotoxic potential ofboric acid in the presence of varying concentrations of zincin vitro. The concentration of boric acid used in this study has been shown in previous EST tests to cause a 50% inhibition of differentiation of D3 cells into contracting myocardial cells (Genschow et al. 2004). The set concentration of boric acid tested was 150 µg/ml with concentrations of zinc chloride of 0, 1.04, 2.08, 10.42, 20.83, 41.67, 83.33 and 125 µg ZnCl/ml, equivalent to 0, 0.5, 1, 5, 10, 20, 40 and 60 µg Zn/ml. No greater sensitivity of embryonic stem cells compared to fully differentiated cells was observed and no concern for in vivo embryotoxicity is triggered for boric acid at various concentrations of zinc. A reduction in the boric acid inhibition of differentiation of D3 embryonic stem cells was observed with increasing concentrations of zinc (Figure 4, see also Appendix G) (Hofman-Huther 2013).

An Oral (Gavage) Prenatal Developmental Toxicity Study of Zinc Borate in Rats

The potential of zinc borate for developmental toxicity was evaluated based on the results of the Developmental Toxicity Test (Edwards 2014). Three treatment groups of 25 Sprague Dawley rats were administered the test article via oral gavage at dose levels of 100, 125 or 150 mg/kg/day. No test substance-related visceral developmental variations were noted in the 100, 125, and 150 mg/kg/day groups. There were no test substance-related skeletal malformations noted for foetuses in the test substance-treated groups. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Higher mean litter proportions of reduced ossification of the 13th rib(s) and sternebra(e) nos. 5 and/or 6 unossified were noted in the 100, 125, and 150 mg/kg/day groups compared to the control group. These findings were considered secondary to the reduced foetal weights noted in these groups. In addition, higher mean litter proportions of 7th cervical ribs and lower mean litter proportions of 14th rudimentary rib(s) were noted in the 100, 125, and 150 mg/kg/day groups and higher mean litter proportions of 25 presacral vertebrae were noted in the 125 and 150 mg/kg/day groups compared to the control group. No test substance-related foetal malformations were observed in the test substance-treated groups. Based on these results, a dosage level of 150 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity and a dosage level of <100 mg/kg/day was considered to be the NOAEL for embryo/foetal development when zinc borate was administered orally by gavage to inbred Crl: CD(SD) rats.

These data show that humans are likely less sensitive to the reproductive and developmental effects of boric acid than laboratory animals due to the comparative high zinc stores in target tissues in humans compared to laboratory animals.

Therefore, based on a total weight of evidence, Category 2 H361d: suspected human reproductive toxicant, suspected of damaging the unborn child is considered the appropriate classification. Extensive evaluations of sperm parameters in highly exposed workers have demonstrated no effects on male fertility. While no developmental effects have been seen in highly exposed populations, epidemiological studies of developmental effects are not as robust as the fertility studies, warranting the Category 2 H361d.

 

Specific Concentration Limit

Boric oxide was formally classified under the 30th ATP to the Dangerous Substances Directive (67/548/EEC) as a Reproductive toxicant Category 2 with a Specific Concentration Limit of 3.1 %. The classification was transferred to the CLP Regulation under the 1st ATP, where the classified category was aligned with the GHS (Category 1B) and the Specific Concentration Limit (SCL) was maintained. Although the ECHA CLP guidance includes setting concentration limits for reproductive toxicants, the current SCL for boric oxide was agreed by the ECB Technical Committee on Classification & Labelling and accordingly became law when the 30th ATP entered into force.