Triboron trimethyl hexaoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found in the body fluids and tissues following absorption by any route of administration. Therefore, an assessment of reproductive toxicity potential was conducted taking account of the hydrolysis breakdown products of TMBX.

No impairment of fertility and reproductive performance was found in male and female rats (parent and daughter generations) exposed to methanol (NEDO, 1987).

In a two-generation reproduction study, rats were exposed to methanol by inhalation for 19-20 hours/day (NEDO, 1987). No treatment-related alterations in general observations and reproductive parameters were found. None of the fertility indices including sexual cycle, days needed for insemination, insemination rate and pregnancy rate showed statistically significant differences. There were no differences for body weight, food consumption and water consumption during gestation and lactation period, either. No abnormalities were observed in findings on delivery and nursing behaviour and necropsy data of F0 animals. In the F1 and F2 progeny (both sexes), no histological changes and no effects on testes or ovaries were reported.

In a one-generation reproduction study in monkeys (Burbacher et al., 1999), adult female monkeys were exposed to methanol vapour (2.5 hours/day; 0, 200, 600, 1800 ppm) during prebreeding, breeding and pregnancy. No signs of overt maternal toxicity were noted during the study in any of the dose groups. Methanol exposure had no effects on the tested reproductive performance, including menstrual cycles, conception rate, and live-birth delivery rate. However, all methanol-exposed animals had a decrease of about 6 to 8 days in duration of pregnancy compared to control animals. It is not clear whether this decrease in duration of pregnancy was related to methanol exposure, since there was no dose-response and no differences among offspring groups in body weight, size or other physical parameters (head size, crown rump length). Moreover, the duration of pregnancy was within the reported normal range for this species. Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months. However, results of infant assessments during the first 9 months of life were confounded by the normal variance and the low number of animals. The NOAEC for reproductive effects can be determined to be at the highest concentration tested of 2.39 mg/L (1800 ppm).

Based on major species differences between humans and rodents (metabolic pathway/enzymes, mode of action, toxicokinetics), considering the overall weight of evidence, and in line with the evaluation of reproductive toxicity provided by the Committee for Risk Assessment (RAC, 2014), methanol does not appear to be toxic to reproduction.

Boric acid is classified under the 1st ATP to 1272/2008/EC as Repr. 1B; H360FD. 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. A multigeneration study in the rat (Weir, 1966) gave a NOAEL for fertility in males of 17.5 mg B/kg/day.  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; the effects to female fertility were inconclusive at non-maternally toxic doses.

Based on the most conservative NOAEL for the hydrolysis products of 17.5 mg B/kg bw day, an equivalent NOAEL for TMBX of 93.73 mg/kg bw/day can be derived. This value is taken as the point of departure for fertility effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

three-generation reproductive 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: Comparable to guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

not applicable

GLP compliance:

no

Remarks:

Study pre-dates GLP

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

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

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

Details on mating procedure:

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

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

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

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

Frequency of treatment:

Daily

Details on study schedule:

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

Remarks:

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

No. of animals per sex per dose:

8 males and 16 females per group

Control animals:

yes, plain diet

Details on study design:

- Rationale for animal assignment: By stratified randomisation

Positive control:

No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

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

Litter observations:

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

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

No data

Statistics:

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

Reproductive indices:

No data

Offspring viability indices:

No data

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

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

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

Dose descriptor:

LOAEL

Effect level:

336 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

LOAEL

Effect level:

58.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

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

Clinical signs:

effects observed, treatment-related

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Description (incidence and severity):

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

Histopathological findings:

not examined

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

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

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

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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Equivalent to 350 ppm boron in the diet.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

17.5 mg/kg bw/day

Based on:

element

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

Table for reproductive toxicity:

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

Conclusions:

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

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedures in accordance with accepted standard methods, well documented, limitation due to low number of animals.

Principles of method if other than guideline:

One generation reproduction toxicity study: Adult female monkeys were exposed to methanol vapour daily during prebreeding, breeding and pregnancy.

GLP compliance:

not specified

Limit test:

no

Species:

monkey

Strain:

Macaca fascicularis

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Cohort 1
- Source: all feral born
- Age at assignment to project: 5.5-11 years old (estimated on the basis of dental records)
- Weight at assignment to project: 2.3-3.7 kg
Cohort 2
- Source: feral born (n=15), colony born (n=9, Texas Primate Center, Charles River Primates, CV Primates or Johns Hopkins University)
- Age at assignment to project: 5-13 years old
- Weight at assignment to project: 2.2-5.7 kg
Both cohorts
- Fasting period before study:
- Housing: Individual (social contact through wire mesh)
- Diet: Purina Laboratory Fiber-Plus® Monkey Diet, once per day in the afternoon
- Water: ad libitum
- Acclimation period: The females were transferred to and from the laboratory (inhalation chamber) in a transfer cage on a daily basis.

The four adult males were feral-born with age estimates between 10 and 12 years. The males weighed between 5 and 7.6 kg, and each had sired an offspring during the project.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: Inhalation chamber housing 1 animal in a cage (47, 61, 80 cm (w, h, d))
- Source and rate of air: Dayton Model 5K901C blower (Dayton Corporation, Moraine, OH), 420 L/min

TEST ATMOSPHERE
Methanol vapour was generated by passing compressed air through gas dispersion bottles filled with methanol. The methanol was heated by placing the bottles in a water bath set at a temperature of approximately 36 °C. The methanol vapour was delivered to the chamber via insulated polypropylene tubing that ran from the bottles to the vapour inlet port of each chamber.

ANALYSIS OF METHANOL AND CARBON DIOXIDE CONCENTRATIONS
Methanol, carbon dioxide, and dew point were measured by withdrawal of an air sample through a polypropylene tube located in the chamber at a level 5 cm above the monkey cage. The air sample was drawn at a rate of approximately 1.5 L/minute. Methanol and carbon dioxide were measured by a General Analysis Corporation (Norwalk, CT) infrared analyzer. Dew point was measured by a General Eastern Instruments (Woburn, MA) hygrometer, and chamber temperature was measured via resistance temperature detectors placed in each chamber. Dew point and temperature were used to calculate the relative humidity(RH) of each chamber.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 4 hours each day on the 11th, 12th and 13th day of the menstrual cycle after exhibiting a minimum of 7 menstrual cycles (3 cycles prior to methanol exposure and 4 cycles after initial exposure)
- Proof of pregnancy: blood progesterone analysis (pregnancy was confirmed by 18 to 20 days of gestation)
- Further matings after two unsuccessful attempts: yes (for 3 additional days, if necessary, a 3rd and 4th breeding took place for 5 consecutive days (days 10 through 14 of cycle)) - always using the same male

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Average chamber concentrations obtained for 11 samples taken from 14 minutes after onset of methanol flow until 6 minutes prior to offset of methanol flow were all within 5 % of the target concentrations.

Duration of treatment / exposure:

During prebreeding: approximately 120 days; during breeding: approximately 70 days; during pregnancy: approximately 165 days.

Frequency of treatment:

Daily for 2.5 hours (7 days/week) before breeding, during breeding and during pregnancy.
At the end of each 2-hour exposure, the animals remained in the chamber for another 30 minutes while the methanol dissipated.

Details on study schedule:

18-November 1992 to 17-December- 1994 (Cohort 1)
23-November-1994 to 07-November-1996 (Cohort 2)

Remarks:

Doses / Concentrations:
0, 0.27, 0.8, 2.39 mg/L (corresponding to 0, 200, 600, 1800 ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

11-12 adult females

Control animals:

yes

Details on study design:

- Dose selection rationale: The target air concentrations were chosen to provide a range of blood methanol concentrations from just above background to just below that reported to cause nonlinear clearance kinetics in primates (Horton et al., 1992).

The two-cohort study design utilized 48 adult females (24 females/cohort), 4 adult males (2 males/cohort), and their offspring. This design minimized the number of subjects tested simultaneously, yet achieved a sufficient sample size to detect subtle changes. For each cohort, adult females were initially separated into 6 groups, with 4 animals per group based on known or estimated age, size, and colony parity. Females from each of the 6 groups were then randomly assigned to one of four methanol-exposure groups.


Reference:

Horton VL, Higuchi MA, Rickert DE (1992). Physiologically based pharmacokinetic model for methanol in rats, monkeys and humans. Toxicol Appl Pharmacol 117: 26–36.

Parental animals: Observations and examinations:

MATERNAL HEALTH ASSESSMENTS

- BODY WEIGHT
- Time schedule: weekly
Females were weighed while being transferred from the inhalation chamber to their homecages.

- CAGESIDE GENERAL OBSERVATIONS
- Time schedule: daily
Each female was observed for signs of lethargy, uncoordinated motor movements (staggering or clumsiness) and laboured or irregular respiration approximately 5 minutes after return to the homecage.

- CLINICAL OBSERVATIONS
- Time schedule: daily
Visual function was assessed by observing whether or not the female could visually orient to and/or follow a syringe filled with apple juice. Fine motor coordination was assessed by observing whether or not the female could reach for and pick up a small piece of fruit using only her thumb and index finger.

- HEALTH CHECK
- Time schedule: daily
Animals were observed for signs of diarrhoea, and medications were administered/recorded.


MATERNAL REPRODUCTIVE ASSESSMENTS
Specific aim of the study was addressed by examining 5 factors in time-mated females: menstrual cycles; frequency of conception; frequency of complications during pregnancy, labour and delivery; duration of pregnancy; and frequency of live births.

- MENSTRUAL CYCLES
- Time schedule: daily
See "Estrous cyclicity" below.

- TIMED MATINGS
See "Details on mating procedure" above.

- PREGNANCY OBSERVATIONS AND DELIVERY EXAMINATIONS
- Time schedule: during the last month of pregnancy (every half hour from 8 p.m. to 6 a.m.)
Females were observed for signs of labour via infrared cameras. Immediately after delivery of an infant, the female was sedated, and the infant was separated from the mother and placed in an isolette. Maternal weights were recorded, and the mother was returned to her home cage for observation while she remained sedated.

Oestrous cyclicity (parental animals):

The onset and duration of menstruation was assessed using a noninvasive observational method to detect menstrual bleeding: the females were trained to present their perinea to an observer for visual evaluation.

Sperm parameters (parental animals):

No adult male monkeys were observed.

Litter observations:

For postnatal developmental evaluation, 8 to 9 infants were available per group, in total 34 infants, among them 26 in-utero treated offsprings.
The birth weight, crown–rump length, and head size of all infants were obtained immediately following delivery. Other infant assessment procedures were also performed at delivery. The results of these assessments on offspring are described in Part II (please refer to section 7.8.2).

Statistics:

Statistical analyses were performed using Systat, SAS, or Splus.Basic hypotheses were developed for the maternal health/reproductive effects part of the study:
3. There will be no significant differences across the methanol exposure groups in overt signs of maternal toxicity.
4. There will be no significant differences across the methanol exposure groups in signs of toxic effects on maternal reproduction.

In addition to these hypotheses, statistical analyses of maternal characteristics (age, weight, crown–rump length, gravidity, parity) at the outset of the study were performed to examine the results of random assignment of adult females to the 4 exposure groups. The general approach to testing the hypotheses was to first assess whether an exposure effect existed, both globally and specifically. A global F test (or equivalent) was used for assessing whether there were detectable differences among the 4 exposure groups. Because this test has less power than specific alternatives, a no exposure–effect hypothesis was also examined that compared the control group with the combination of all methanol-exposure groups. The control group was also compared with each methanol exposure group using pairwise comparisons. Finally, the impact of controlling for cohort was assessed in mean models.

To test Hypothesis 3, four separate procedures were used to detect overt signs of maternal toxicity. Due to the low number of positive responses, descriptive analyses were performed.
To test Hypothesis 4, the following measures of toxic effects on maternal reproduction were used:
(a) menstrual-cycle length
(b) rate of conception
(c) weight gain during pregnancy
(d) frequency of pregnancy and delivery complications
(e) pregnancy duration
(f) frequency of live-birth deliveries
(g) offspring birth size (weight, crown–rump length, head circumference, length, and width)
Repeated measures ANOVA models, one-way ANOVA models and Fisher's exact test were used to assess statistical differences.

Reproductive indices:

Conception rate, live birth delivery rate

Offspring viability indices:

The birth weight, crown-rump length, and head size of all infants were obtained immediately following delivery.

Methanol exposure had no effect on most measures of reproductive performance, including menstrual cycles, conception rate, and live-birth delivery rate. However, all methanol-exposed animals had a decrease of about 6 to 8 days in duration of pregnancy compared to control animals.

MATERNAL HEALTH ASSESSMENTS
(Methanol exposure and maternal toxicity (Hypothesis 3)):

- BODY WEIGHT
The weights of all females were quite stable during the study. The mean weight for each of the 4 methanol exposure groups during the baseline period and through breeding was approximately 3.5 kg. The mean weight at conception for the females in the 4 exposure groups was between 3.2 and 3.7 kg. Mean weight gain during pregnancy varied from 1.3 to 1.8 kg across all exposure groups. Weight gain during pregnancy was calculated for each female and used in ANOVA models to test for differences across the methanol exposure groups. The results did not indicate a significant methanol exposure effect on maternal weight gain during pregnancy (p > 0.12, all tests).

- CAGESIDE GENERAL OBSERVATIONS
There was no overt toxicity in adult females from any of the 4 methanol exposure groups. Lethargy, uncoordinated motor movements, and laboured respiration were not observed during the study.

- CLINICAL OBSERVATIONS
The results of 50 (Cohort 2) to 100 (Cohort 1) clinical observations did not indicate the presence of overt toxicity in the adult females. During the entire study, only 6 females failed to respond to the visual task (3 control females and 3 methanol exposed females). Of the 46 females observed, 23 failed the motor coordination task during the study. Of these 23 females, 15 failed it 3 times or fewer. Of the remaining 8 females, who failed the task 5 to 13 times, 4 were from the control group, 1 was from the 200 ppm exposure group, and 3 were from the 600 ppm exposure group. All of these females failed the task during the baseline period as well as during methanol exposure, and none exhibited a pattern of responses indicative of fine-motor incoordination due to methanol exposure.

- HEALTH CHECK
The number of females who became ill and required medication was quite low, and unrelated to dose.


MATERNAL REPRODUCTIVE ASSESSMENTS
(Methanol exposure and reproductive toxicity (Hypothesis 4))

- MENSTRUAL CYCLES
All females exhibited 3 menstrual cycles before methanol exposure, 1 cycle during the period in which exposure was started, and 3 cycles after exposure was started. One female exhibited an abnormal cycle length of 88 days prior to methanol exposure. This cycle was not included in the analysis. All females exhibited at least 4 normal cycles (>20 days and 50 days) prior to breeding.
The results of the ANOVA models did not indicate significant differences in the lengths of menstrual cycle of females across the 4 methanol exposure groups during the baseline period (p > 0.12, all tests). There was no statistically significant methanol exposure effect on cycle lengths in the females (p = 0.45). The duration of menstrual cycle remained stable at approximately 30 days.

- TIMED MATINGS
The frequency of conception was approximately the same across the 4 exposure groups (82 % in the control group, 75 % at 200 ppm, 82 % at 600 ppm, and 83 % at 1800 ppm). Conception frequencies were not affected by methanol exposure (p = 1.0).

- PREGNANCY OBSERVATIONS AND DELIVERY EXAMINATIONS
A total of 37 infants were delivered from the 46 females. Two females delivered stillborn infants, 1 infant in the control group and 1 infant at 600 ppm. One female at 1800 ppm required a Caesarean (C) section to deliver a dead fetus.
The rate of complications during pregnancy or labour and delivery were 22 % for the control females and the 200 ppm females, 33 % for the 600 ppm females, and 30 % for the 1800 ppm females. No significant differences across methanol exposure groups were found (p = 1.0). The live-birth delivery rates were between 90 and 100 % for all 4 exposure groups. There were no sire related effects on pregnancy length. The results of the ANOVA model indicated a significant effect on pregnancy length due to methanol exposure (p = 0.03). Post hoc testing indicated that each of the 3 methanol-exposed groups had significantly shorter durations of pregnancy than did the control group (p < 0.04, all tests).

Dose descriptor:

NOAEC

Effect level:

2.39 mg/L air (nominal)

Sex:

female

Basis for effect level:

other: reproductive performance

MATERNAL REPRODUCTIVE ASSESSMENTS
(Methanol exposure and reproductive toxicity (Hypothesis 4))

- PREGNANCY OBSERVATIONS AND DELIVERY EXAMINATIONS
The offspring characteristics analyzed were birth weight, crown–rump length, head circumference, head length, and head width. The results of the ANOVA models did not indicate a significant effect of methanol exposure on offspring size at birth (p > 0.24, all tests). ANOVA models controlling for cohort differences, however, indicated a significant methanol-exposure-group-by-cohort interaction for birth weight (p < 0.002) and head circumference (p < 0.03).

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

2.39 mg/L air (nominal)

Sex:

not specified

Basis for effect level:

other: growth and physical development of the offsprings

Reproductive effects observed:

not specified

Exposure to methanol at concentrations of up to 1800 ppm for over 1 year did not produce overt signs of toxicity (motor incoordination, blindness, and/or respiratory effects) in adult female nonhuman primates. Chronic methanol exposure did not interfere with the menstrual cycle or the ability of females to conceive. The timed-mating procedures used (3 matings/day between days 11 and 13 of the menstrual cycle) typically produce close to 100 % conception rates in normal groups of M. fascicularis females (Mahoney, 1975). The overall conception rate for this study was lower than expected, at 80 %. This was due to a sire effect: 1 of the males in Cohort 2 successfully impregnated only 4 females.

There was no pregnancy-induced folate deficiency. Fetal and newborn mortality frequencies were low for all of the exposure groups. One female at 1800 ppm had to be C-sectioned to deliver a dead fetus, and 2 females (1 control infant, 1 infant at 600 ppm) vaginally delivered full-term stillborn infants. The autopsy on the fetus delivered by C-section indicated the presence of hydrocephalus with significant autolysis in all of the major organs. Autopsies on the 2 stillborn infants indicated that the lungs were not inflated and that they had died close to or during delivery. No malformations were observed, and the cause of death for both infants was asphyxiation.

Two methanol-exposed females each at 200 ppm and 600 ppm were C-sectioned following observations of uterine bleeding without productive labour, presumably due to placental detachment. All 4 infants were delivered alive and without complications. Given the small number of animals exhibiting this condition and the lack of a response at the highest exposure concentration, conclusions concerning methanol exposure as a causative factor in uterine bleeding are not warranted.

It was not clear whether the decrease of about 6 to 8 days in duration of pregnancy noted as compared to controls was related to methanol exposure, since there was no dose-response and no differences among offspring groups in body weight or other physical parameters.

Although the average gestation period of the methanol exposed offspring was significantly shorter than that of the controls, methanol exposure did not affect the size of the offspring at birth. The average birth weight, crown–rump length, and head size of infants in the methanol exposure groups were comparable to those of the control infants. These results do not indicate that reduced offspring size at birth is associated with methanol exposure concentrations insufficient to cause overt maternal toxicity.

Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months.

Reference:

Mahoney C. (1975.) Practical aspects of determining early pregnancy, stage of foetal development, and imminent parturition in the monkey (Macaca fascicularis). Lab Anim 6: 261–274.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

93.73 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The most reliable comparable to guideline study with acceptable restrictions.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

TMBX is hydrolytically unstable and breaks down to form methanol and boric acid in the presence of water, these species can be expected to be found in the body fluids and tissues following absorption by any route of administration. Therefore, an assessment of developmental toxicity potential was conducted taking account of the hydrolysis breakdown products of TMBX.

Based on the available evidence, humans are not susceptible to the methanol-related developmental toxicity observed in rats and mice, due to the substantial species differences in metabolism. Different enzymes involved in methanol metabolism provide the basis

for the kinetic differences between rodents and humans, which result in the formation of different metabolites at different metabolic rates. Moreover, it is clear from available animal data that, based on differences in metabolism and the formation/accumulation of formic acid in humans, which leads to maternal toxicity at much lower concentrations, the developmental effects observed in rats and mice in the absence of maternal toxicity are not relevant to humans. Methanol is presented in the ECHA Guidance on CLP (2015) as an example for not using rodent toxicity data to classify methanol for acute toxicity and specific organ toxicity on the basis of the non-relevance of rodent toxicity data to humans. This is due to species differences between humans and rodents, rendering the rodent data on methanol irrelevant to humans. The same approach should be applied for developmental toxicity assessment. Methanol has a uniquely high acute toxicity for humans with target organ toxicity for the ophthalmic nerve, which is different to toxicity seen in rodents. There is a large database on the toxicity of methanol and more recent data further support the previous EU decision not to classify methanol for developmental toxicity.

Developmental effects from borate exposure 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 BMDL05 of 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.

BAsed on the BMDL05 for Boric acid of 10.3 mg B/kg bw/day, an equivalent BMBL05 for TMBX of 55.2 mg/kg bw/day can be derived.

Link to relevant study records

Reference

Reference 1

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. Read-across is justified on the following basis: The family of zinc borates that include Zinc Borate 500, Zinc Borate 2335 and Zinc Borate 415 (also known as Zinc Borate 411). Zinc borate 500 is anhydrous Zinc Borate 2335 and Zinc Borate 415 has different zinc to boron ratio. Zinc borate 2335 (in common with other zinc borates such as Zinc borate 415 and 500) breaks down to Zinc Hydroxide (via Zinc oxide) and Boric Acid, therefore the family of zinc borates shares the same toxicological properties. Zinc borates are sparingly soluble salts. Hydrolysis under high dilution conditions leads to zinc hydroxide via zinc oxide and boric acid formation. Zinc hydroxide and zinc oxide solubility is low under neutral and basic conditions. This leads to a situation where zinc borate hydrolyses to zinc hydroxide, zinc oxide and boric acid at neutral pH quicker than it solubilises. Therefore, it can be assumed that at physiological conditions and neutral and lower pH zinc borate will be hydrolysed to boric acid, zinc oxide and zinc hydroxide. Hydrolysis and the rate of hydrolysis depend on the initial loading and time. At a loading of 5% (5g/100ml) zinc borate hydrolysis equilibrium may take 1-2 months, while at 1 g/l hydrolysis is complete after 5 days. At 50 mg/l hydrolysis and solubility is complete (Schubert et al., 2003). At pH 4 hydrolysis is complete. Zinc Borate 2335 breaks down as follows: 2ZnO • 3B2O3 •3.5H2O + 3.5H2O + 4H+ ↔ 6H3BO3 + 2Zn2+ 2Zn2+ + 4OH- ↔ 2Zn(OH)2 ____________________________________________________________ Overall equation 2ZnO • 3B2O3 •3.5H2O + 7.5H2O ↔ 2Zn(OH)2 + 6H3BO3 The relative zinc oxide and boric oxide % are as follows: Zinc borate 2335:zinc oxide = 37.45% (30.09% Zn) B2O3 = 48.05% (14.94% B) Water 14.5% Zinc borate 415: zinc oxide = 78.79%; (63.31% Zn) B2O3 = 16.85% (5.23% B) Water 4.36% Zinc borate, anhydrous: Zinc oxide = 45 % B2O3= 55% (17.1 % B)

Qualifier:

no guideline required

Principles of method if other than guideline:

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

GLP compliance:

not specified

Remarks:

not applicable (it is a publication)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Route of administration:

oral: feed

Vehicle:

not specified

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

20 days

Frequency of treatment:

continuously in diet

Remarks:

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

Remarks:

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

No. of animals per sex per dose:

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

Control animals:

yes, plain diet

Details on study design:

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

Dose descriptor:

BMD:

Effect level:

59 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

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

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.

Executive summary:

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

Reference 2

Endpoint:

developmental toxicity

Type of information:

other: Review publication

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

BMDL05

55.2 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The study meets acceptable scientific standards with acceptable restrictions.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Boric acid is classified under the 1^stATP to 1272/2008/EC as Repr. 1B; H360FD. A recent Opinion of the ECHA Committee for Risk Assessment (2104) has confirmed the classification; the specific concentration limit of 5.5% w/w was unchanged.

Based upon the products of hydrolysis of TMBX, resulting in a mixture containing approximately 61.8% w/w Boric acid and 32% w/w Methanol, TMBX should be classified as Reproductive Toxicant Category 1B (H360FD) in accordance with the classificaiton criteria of 1272/2008/EC.