Pentaboron sodium octaoxide

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1963/09/23 - 1965/06/28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1966

Report date:

1966

Reference 2

Reference Type:

publication

Title:

Toxicologic studies on borax and boric acid.

Author:

Weir RJ & Fisher RS

Year:

1972

Bibliographic source:

Toxicology and Applied Pharmacology 23: 351 - 364.

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Boric acid

EC Number:

233-139-2

EC Name:

Boric acid

Cas Number:

10043-35-3

Molecular formula:

H3BO3

IUPAC Name:

Boric acid

Details on test material:

- Name of test material: Boric acid
- Physical state: Fine white powder without odour
- Analytical purity: > 99 %
- Stability under test conditions: Stable

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

34 mg/kg bw/day (nominal)

Remarks:

equivalent to 5.9 mg B/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

equivalent to 17.5 mg B/kg bw/day

Doses / concentrations 4

Dose / conc.:

336 mg/kg bw/day (nominal)

Remarks:

equivalent to 58.5 mg B/kg bw/day

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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.

3

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).

4

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

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.

Effect levels (F1)

1

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.

2

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.

Results: F2 generation

Effect levels (F2)

1

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.

2

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.