Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions.
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
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
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Equivalent to 350 ppm boron in the diet.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
17.5 mg/kg bw/day
Based on:
element
Sex:
male/female
Basis for effect level:
other: No adverse effects in mid and low dose groups in any generation.
Reproductive effects observed:
not specified

Table for reproductive toxicity:

Parameter

 

control

low dose

medium dose

High dose

 

Generation

m

f

m

f

m

f

m

f

 

 

Mortality

incidence

P

0

1

0

0

0

0

0

0

 

 

 

 

F1

0

1

0

0

0

0

0

0

 

 

 

 

F2

0

0

0

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Food consumption

% of control

not affected

 

 

 

 

 

 

 

 

 

 

Body weight gain

% of control

 

-

-

-

-

-

-

¯

¯

 

 

Clinical Observations

specify effects

Incidence

 

-

-

-

-

-

-

+

  +

 

 

Organ weights

% of control

only effect noted was increase in absolute wt. of thyroid in low dose group and relative thyroid wt. in low and mid dose groups (not thought to biologically significant)

Pathology

 

 

 

 

 

 

 

 

 

 

 

 

Histopathologic examination

specify effects

Incidence

Evidence of testis atrophy in high dose males of P0 generation.

Evidence in ovary of reduced ovulation in high dose females.

Reproductive Performance

 

P0 to F1a

F1b to F2b

F2b to F3b

 

cont

low

mid

high

cont

low

mid

cont

low

mid

 

Mating index: (No. pregnant/No. mated)

%

62

88

81

0

80

94

94

69

94

94

 

Fertility index: No. litters born/No. Pregnant

%

100

100

100

-

100

100

100

91

100

100

 

Number of implantation sites

Mean

 

 

 

 

 

 

 

 

 

 

 

Duration of pregnancy

Mean

 

 

 

 

 

 

 

 

 

 

 

Birth index

 

 

 

 

 

 

 

 

 

 

 

 

Live birth index: No.pups alive/No. born

%

98

96

97

 

99

99

98

100

99

99

 

Gestation index

 

 

 

 

 

 

 

 

 

 

 

 

Litter size

Mean

12

11

11

 

12

13

12

12

13

11

 

Litter weight

Mean

 

 

 

 

 

 

 

 

 

 

 

Pup weight at 24h (g)

Mean

7.0

7.2

6.7

 

6.4

6.5

6.7

6.0

7.0

7.0

 

Sex ratio

Male/female

6/6

6/5

5/6

 

6/6

7/6

6/6

6/6

7/6

6/5

 

Survival index

 

 

 

 

 

 

 

 

 

 

 

 

Viability index

 

 

 

 

 

 

 

 

 

 

 

 

Lactation index: Pup wt. at weaning

 

55

50

52

 

56

53

51

48

51

55

 

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

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.


Short description of key information:
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 multigeneration study in the rat (Weir, 1966) gave a NOAEL for fertility in males of 17.5 mg B/kg/day, which corresponds to 168.18 mg C3H9BO3/kg bw / day.

Justification for selection of Effect on fertility via oral route:
weight of evidence

Effects on developmental toxicity

Description of key information
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).
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets acceptable scientific standards with acceptable restrictions.
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
Dose descriptor:
BMD:
Effect level:
10.3 mg/kg bw/day
Based on:
element
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
BMDL05
98.98 mg/kg bw/day
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

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.

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).

Justification for classification or non-classification

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.