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Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects expected for boron if tested at a limit dose of 1000 mg/kg/day. The hazard conclusions regarding effects on fertility and pre-natal development are based on a weight-of-evidence assessment and waiving. For a detailed explanation see "Additional information (fertility)".

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

1. Approach to address the standard information requirements for reproductive (fertility) and pre-natal developmental toxicity:

In accordance with REACH Annex IX, 8.7, the standard information requirements for Boron, which is registered for the 100-1000 tpy tonnage band, include an Extended One Generation Reproductive Toxicity Study (EOGRTS) and a Prenatal Developmental Toxicity Study in one species.

Both these studies are waived for boron, based on REACH Annex IX, 8.7, column 2 reasons (“low toxicological activity, no systemic absorption, no significant human exposure”). Extensive justification for this waiving is provided in the corresponding endpoint study records in the technical IUCLID dossier, section 7.8.1 “waiving annex IX (EOGRTS)” and 7.8.2 “waiving_annex IX (PNDT,rat)”.

For essentially the same reasons, a sub-chronic 90-day toxicity study with boron is also not required, see the very extensive waiving justification provided in the technical dossier IUCLID section 7.5.1. This document also discusses in detail the most relevant route of exposure to boron, which is the oral route.

When both EOGRTS and PNDT studies are waived - according to the IUCLID validation assistant (IUCLID 6, 4.14.1, build: 22/04/2020 12:18) - the data requirement for a 28-day screening test for reproductive/developmental toxicity (i.e. OECD TG 422) shall be addressed (REACH Annex VIII, 8.7.1).

Whereas the same scientific logic (“low toxicological activity, no systemic absorption, no significant human exposure”) applies to a 28-day screening test for reproductive/developmental toxicity, Annex VIII, 8.7, column 2, does not formally provide this reason for waiving, whereas Annex IX does.

Therefore, the data requirement for the 28-day screening test for reproductive/developmental toxicity is formally addressed in this dossier by a weight-of-evidence approach, in accordance with REACH Annex XI, 1.2. For technical dossier completeness an endpoint study record indicated as “weight of evidence” in the “adequacy of study” field has been created for the endpoint “screening for reproductive/developmental toxicity”. This endpoint study record describes the available 28-day study (OECD TG 407), which together with other information leads to the hazard conclusion for reproductive toxicity in a weight-of-evidence approach, see below.

 

2. Overview of key available toxicological and toxicokinetic studies for boron:

The following key toxicological studies using boron (powder) as the test substance are available and described in the respective sections of the dossier and further below in this endpoint summary. No adverse effects are observed in these studies at the respective limit doses.

- acute oral and inhalation toxicity (OECD TG 423/403), see technical dossier section 7.2

- sub-acute (28day) oral toxicity study (OECD TG 407), see technical dossier section 7.5.1 and summary in below.

These, together with toxicokinetic investigations in the 28-days study and a mass balance study (as follows), demonstrate the absence of any significant systemic absorption of boron following oral administration.

- oral “mass balance” and “blood kinetic” studies, see technical dossier section 7.1

 

These “negative” studies are further supported by the absence of any adverse effects in skin and eye irritations studies, in a skin sensitisation study, and in a standard set of in-vitro genotoxicity studies. Negligible solubility/bioaccessibility was also observed in experiments on water solubility and bioelution in artificial gastric juice (study summaries in technical dossier sections 4.8. and 7.1.1).

 

3. Toxicological results from 28-day limit test

A 28-day repeat dose toxicity limit test in rats was conducted (Leuschner, 2013) to assess the effect of elemental boron on rats following repeated oral administration. The study was conducted according to OECD test guideline 407, and in compliance with GLP.

Male and female rats were administered elemental boron by oral gavage for 28 days at a dose of 1000 mg/kg bw/day in 0.8% aqueous hydroxyl propyl methylcellulose gel. A concurrent control group was administered with untreated vehicle.

No clinical signs of toxicity were observed, and no animals died during the administration period. No changes in bodyweight gains, food consumption, haematology, clinical chemistry, organ weights, macropathology or histopathology were observed which could be attributed to treatment with the test compound. No adverse effects were observed on the male and female reproductive organs.

It is concluded that elemental boron was well tolerated and that no signs of systemic toxicity had been seen in rats when administered at a dose of 1000 mg/kg bw/day for up to 5 weeks. No increase in plasma boron concentrations and only a minor fraction of the total administered boron was collected via urine demonstrates the lack of bioavailability of elemental boron. The no observed adverse effect level (NOAEL) in rats is 1000 mg/kg/day.

See also robust study summary in technical dossier, section 7.5.1.

Reference: Leuschner, J. (2013). 28-day subchronic oral toxicity study of boron, amorphous in rats. Testing laboratory: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, Germany. Report no.: 29178.

 

4. No evidence of systemic absorption of boron

4.1 Toxicokinetic screening data from a 28 day repeated dose oral toxicity study

In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of elemental boron of 1,000 mg/kg bw/day via gavage. Individual urine samples were collected from all animals prior to sacrifice in one cumulated 24-h fraction/animal after the last oral application, and blood samples were collected from each animal upon sacrifice. The plasma and urine samples were analysed for total boron content. No increase whatsoever in boron plasma concentrations of the treated animal in comparison to controls was observed, the (boron in plasma from the control group and dosed group being below detection limit in all samples).

Only a very slight increase in boron concentrations in urine from treated animals was observed. The boron concentration of the 24h-urine samples, collected during the day before final sacrifice, ranged from 1.8 to 6.0 mg boron/L urine (mean: 4.24 ±1.72) and 2.5 to 4.5 mg boron/L urine (mean: 3.24 ±0.83) for the male and female animals of the control group respectively. For the dosed group, the concentrations were 7.6 to 14.8 mg boron/L urine (mean: 12.16 ±2.81) and 5.5 to 11.2 mg boron/L urine (mean: 8.04 ±2.24) for the male and female animals of the dose group respectively.

Following a subtraction of the background urinary boron excretion (control group), and taking into account the excreted urine volume (mean 17.2 mL (m) and 16.8 mL (f)) and the body weight of the animals at the end of the study (mean 348 g (m) and 199 g (f)), the following conclusion can be made:

From a final dose of 1,000 mg/kg boron that the animals received on the last day of the study, only cumulated relative amounts of 0.039 % (m) or 0.040% (f) were found in the terminal 24-h urine collection period.

See also robust study summary in technical dossier, section 7.5.1.

Reference: Leuschner, J. (2013). 28-day subchronic oral toxicity study of boron, amorphous in rats. Testing laboratory: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, Germany. Report no.: 29178.

 

4.2 Supporting comparative Mass-Balance Study

In a comparative mass balance study involving oral dosing of (i) amorphous (elemental) boron and (ii) a soluble borate substance (boric acid), the gastrointestinal absorption as well as urinary and faecal excretion were compared, plus consideration of dietary „background“ intake/excretion via a vehicle-dosed control. For details, please refer to the corresponding robust study summary (7.1.1). In brief, 10 (5m/5f) per group received a single oral dose of 1000 mg/kg elemental boron or 572 mg/kg boric acid (corresponding to 100 mg/kg “boron”). A third group served as vehicle treated control. Animals were individually housed in metabolic cages and daily samples of urine and faeces were collected for three days. All samples were analysed for boron. The averaged “background” excretion via urine and faeces of the control animals was subtracted from the amounts excreted by the dosed animals, and a mass balance was calculated.

Animals that received 100 mg B/kg bw (as boric acid) orally excreted 86.1 % of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals).

The largest fraction (80.7 %) was excreted via urine already within the first 24h. These findings indicate a rapid absorption of boric acid in the gastrointestinal tracts, as well as a rapid renal clearance.

Animals that received 1000 mg B /kg bw (as elemental boron) orally excreted 0.05 % of the administered dose via urine and 99.45% via faeces during the first three days after exposure (mean for 10 animals); the cumulative excretion reached 99.5%.

The largest fraction (93.9 %) was excreted via faeces already within the first 24h with a further 5.1% being excreted via faeces on the second day. Urinary excretion was negligible (only 0.05% of administered dose excreted over three days combined).

At 99.5%, the mass balance is essentially complete and these findings indicate that elemental boron is not absorbed in the gastrointestinal tract of rats to any significant extent, but passes through the animal effectively unchanged.

See also robust study summary in technical dossier, section 7.1.1.

Reference: Leuschner, J. (2015a). Investigations of the mass balance of boron (amorphous) and boric acid in CD rats following a single oral administration. Testing laboratory: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG Redderweg 8 21147 Hamburg Germany. Report no.: 29810.

 

4.3 Supporting relative bioavailability study

A relative bioavailability study involving serum kinetics over a period of 72 hours post administration involving an intravenous dosing of a soluble borate reference substance (boric acid) compared to single oral doses of boric acid and amorphous (elemental) boron. For details, please refer to the corresponding robust study summary (7.1.1). In brief, 20 animals (10m/10f) per group received single doses of (1) 572 mg/kg boric acid intravenously, (2) 572 mg/kg boric acid via oral gavage, and (3) 1000 mg/kg elemental boron via oral gavage.

Blood samples were taken at 0, 0.5, 1, 2, 3, 4, 8, 12, 24, 48 and 72 hours post exposure and blood plasma samples were prepared and analysed for boron. To reduce the stress for the animals, not every animal was sampled at every time-point. Instead, 10 animals (5m/f5) from each group were sampled at every second time-point.

Cmax-levels in plasma of 101.18 µg boron/mL and 95.41 µg boron/mL were noted 0.5 hours after intravenous administration of 572 mg boric acid/kg for the male and female rats on test day 1, respectively. Furthermore, Cmax-levels of 37.92 µg boron/mL and 34.03 µg boron/mL were noted 2 or 1 hours after oral administration of 572 mg boric acid/kg for the male and female rats on test day 1, respectively. Lastly, Cmax-levels of 1.15 µg boron/mL and 0.94 µg boron/mL were noted 2 hours after oral administration of 1000 mg boron, amorphous/kg for the male and female rats on test day 1, respectively. For comparison, the average (n=30) concentration of boron in plasma taken before exposure at t=0 h was (0.097 ± 0.059) µg/mL (min=0.04; max=0.34).

The plasma concentrations declined post dosing with an elimination half-life ranging from 2.73 to 4.91 hours.

A relative bioavailability of 91% was calculated for boric acid following oral administration compared to intravenous administration, and of approximately 0.1% for boron, amorphous (0.09 % in males, 0.10% in females).

See also robust study summary in technical dossier, section 7.1.1.

Reference: Leuschner, J. (2015b). Toxicokinetics of boron (amorphous) and boric acid in CD rats following a single intravenous or oral administration. Testing laboratory: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG Redderweg 8 21147 Hamburg Germany. Report no.: 29811.

 

4.4. Conclusion: no evidence of absorption of boron

When comparing the findings of in-vitro dissolution testing with the in-vivo results, the in-vivo data consistently demonstrate slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

Thus, the oral bioavailability of elemental boron (powder) can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparable results and supported by in-vitro dissolution experiments in pure water and five different artificial physiological fluids. The apparent in-vivo absorption at levels < 0.1 % can be attributed to an extremely thin “oxidised” layer of boric acid that forms on the surface of amorphous boron particles.

A rounded value of 0.05% for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg B/kg bw/d and (ii) a mass balance study involving a single dose of 1,000 mg B/kg bw.

 

5. Weight of evidence approach: OECD TG 422 screening test for reproductive/developmental toxicity study would not show adverse effects.

By comparison with the available OECD TG 407 28-day oral toxicity study, a OECD TG 422 study can further provide “initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition” (quoted from OECD TG 422, section 7). The principles of the two tests differ. Very briefly, male rats are dosed for 28-days in both studies, but the OECD TG 422 study includes dosing of females for up to approximately 63 days (at least 14 days pre-mating, (up to) 14 days mating, through ca. 22 days of gestation and continuing for a further 13 days though lactation). Obviously, the OECD TG 422 study investigates several parameters relating to fertility that are not studied in an OECD TG 407 study and the OECD TG 422 also includes an evaluation of the litter for developmental effects.

However, it has been shown that systemic absorption of elemental boron following oral dosing of rats is insignificant, even at 1000 mg/kg/day for 28 days. Several studies support this conclusion (the 28-day study, the mass balance and blood kinetic studies, as well as in-vitro bioaccessibility and water solubility data). All these studies are described in the respective sections of the dossier (4.8, 7.1.1, 7.5.1), and are also summarised and discussed in the three extensive waiving statements for sub-chronic (90-day) toxicity, EOGRTS and PNDT studies in the technical dossier sections 7.5.1, 7.8.1 and 7.8.2, respectively.

When a chemical/substance is not absorbed systemically, but instead passes the gastrointestinal tract unchanged (excretion via faeces) it cannot have any effects (negative or positive) on reproductive functions/organs, nor reach the developing offspring in the uterus.

It is therefore justified to conclude – based on this weight-of-evidence approach – that an OECD TG 422 study with oral dosing of elemental boron at 1000 mg/kg/day would not produce any adverse effects on neither male rats nor on the dams, and also not on the F1 generation offspring. The same applied to the EOGRTS study and the “full” PNDT study (OECD TG 414).

The overall hazard conclusion for both “fertility” and “prenatal developmental” toxicity is therefore “no hazard identified”, with an unbounded NOAEL of > 1000 mg/kg/day.

 

Effects on developmental toxicity

Description of key information

No adverse effects expected for boron if tested at a limit dose of 1000 mg/kg/day. The hazard conclusions regarding effects on fertility and pre-natal development are based on a weight-of-evidence assessment and waiving. For a detailed explanation see "Additional information (fertility)".

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The overall hazard conclusion for both “fertility” and “prenatal developmental” toxicity is “no hazard identified”, with an unbounded NOAEL of > 1000 mg/kg/day. No hazard classification is required.

Additional information