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Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1966
Report Date:
1966
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 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
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

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 animals)

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
Gross pathological findings:
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)

open allclose all
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

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
Histopathological findings:
not examined

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)

open allclose all
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

Results: F2 generation

Effect levels (F2)

open allclose all
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.

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.