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EC number: 204-468-9 | CAS number: 121-43-7
Table for reproductive toxicity:
% of control
Body weight gain
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)
Evidence of testis atrophy in high dose males of P0 generation.
Evidence in ovary of reduced ovulation in high dose females.
P0 to F1a
F1b to F2b
F2b to F3b
Mating index: (No. pregnant/No. mated)
Fertility index: No. litters born/No. Pregnant
Number of implantation sites
Duration of pregnancy
Live birth index: No.pups alive/No. born
Pup weight at 24h (g)
Lactation index: Pup wt. at weaning
Formally, the REACH Annex XI standard testing regime adaptation options of 1.5 – Grouping of substances and read-across approach and 2. – Testing technically not possible, apply to trimethyl borate: REACH Annex XI lists substances with ‘common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals’, as qualifying for read-across.Additionally, as trimethyl borate will not exist in an organism for longer than one second, it is technically not possible to test its effects.Based on the instant hydrolysis of trimethyl borate to release boric acid (see IUCLID section 5.1.2 and analogue approach rationale in section 13), reproductive toxicity information from boric acid is taken forward for hazard characterisation.
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).
Although boron has been shown to adversely affect male reproduction in laboratory animals, male reproductive effects attributable to boron have not been demonstrated in multiple studies of highly exposed workers. For further information on epidemiologic studies with workers exposed to high concentrations of boron, please refer to chapter 7.10.2 of this dossier and the respective endpoint summary.
The overall evidence on a lack of human relevance of the male fertility findings calls the use of the dose descriptors from the animal studies for hazard characterisation and the resulting classification into question.
Formally, the REACH Annex XI standard testing regime adaptation options of 1.5 – Grouping of substances and read-across approach and 2. – Testing technically not possible, apply to trimethyl borate: REACH Annex XI lists substances with ‘common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals’, as qualifying for read-across. Additionally, as trimethyl borate will not exist in an organism for longer than one second, it is technically not possible to test its effects. Based on the instant hydrolysis of trimethyl borate to release boric acid (see IUCLID section 5.1.2 and analogue approach rationale in section 13), reproductive toxicity information from boric acid is taken forward for hazard characterisation. A benchmark dose of 59 mg boric acid/kg bw/day (10.3 mg B/kg bw/day or 98.98 mg C3H9BO3/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).
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; 85 mg disodium tetraborate decahydrate/kg, 65 mg disodium tetraborate pentahydrate/kg and 44.7 mg disodium tetraborate anhydrous/kg.
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).
Boric acid is classified under the 1stATP to CLP as Repr. 1B; H360FD. Trimethyl borate will be formally classified accordingly.
The text of the 30th ATP as published in the EU Official Journal, 15 September 2008 stated that“The classification and labelling of the substances listed in this Directive should be reviewed if new scientific knowledge becomes available. In this respect, considering recent preliminary, partial and not peer-reviewed information submitted by industry, special attention should be paid to further results of epidemiological studies on the Borates concerned by this Directive including the ongoing study conducted in…”
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). Not only are these the most exposed workers, but the Chinese and Turkish worker studies are the most sensitive studies that have been carried out as semen analysis was performed, a very sensitive detection system for testicular damage. 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). However, studies of human developmental effects are not as robust as the studies of male reproduction because of developmental ascertainment issues.
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 the 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 whystudies of highly exposed boron industry workers have shown no adverse effects anddemonstrates 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.
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.
Therefore, based on a total weight of evidence, Category 2 H361d: suspected human reproductive toxicant, suspected of damaging the unborn child could also be considered an 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. However, boric acid is officially classified under the 1stATP to CLP as Repr. 1B; H360FD. Trimethyl borate will be formally classified accordingly.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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