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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Toxicologic Studies on Borax and Boric Acid
Author:
R.J. Weir, Jr. and R.S. Fisher
Year:
1972
Bibliographic source:
Toxicology and Applied Pharmacology 23, 351-364

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Three Generation study with two litters per generation.
- Short description of test conditions: Cesarean derived rats were randomized in groups of 8 males and 16 females. The animals received borax or boric acid at 117, 350 and 1170 ppm as boron equivalent in the diet. Prior to initiation of the first breeding phase, the animals were maintained in individual cages and fed their respective diets for 14 weeks. After the 14 week feeding period, 1 male and 2 females were placed in each breeding cage. At 24 hours after birth, the litters were reduced to a maximum of 8 progeny to be raised. The first filial generation (F1a) was carried through weaning and discarded. The parental generation (P1) was rebred to produce their second litter (F1b). At the time of weaning, 16 females and 8 males each from the control and test groups were selected at random and designated the second parental generation (P2) for continuation of the reproduction study. These animals were bred to produce the F2a and F2b litters as before. The F2b litter became the P3 generation and were bred to produce the F3a and F3b litters.
- Parameters analysed / observed: Body weight and food consumption were recorded weekly. With the exception of the P1, P2 and P3 control and test groups, necropsies were performed on all rats.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Caesarean-derived rats from Charles River, USA
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: not specified
- Weight at study initiation: 110 - 150 g
- Fasting period before study: no
- Housing: individually except during mating, during mating 1 male and 2 females per cage
- Diet: Purina Laboratory Chow
- Water: not specified
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not specified
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

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 Purina Laboratory Chow: The test material was incorporated into the basal diet on a weight/weight basis and thoroughly mixed to provide the desired dietary levels.
Details on mating procedure:
- M/F ratio per cage: 1:2
- Length of cohabitation: not specified
- 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.
Analytical verification of doses or concentrations:
not specified
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 until sacrifice of the F1- and F2-generations.
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters. : not specified
- Selection of parents from F1 generation at the time of weaning.
- Age at mating of the mated animals in the study: not specified
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.
Doses / concentrationsopen allclose all
Dose / conc.:
117 ppm (nominal)
Remarks:
equivalent to 5.9 mg boron/kg bw/day
Dose / conc.:
350 ppm (nominal)
Remarks:
equivalent to 17.5 mg boron/kg bw/day
Dose / conc.:
1 170 ppm (nominal)
Remarks:
equivalent to 58.5 mg boron/kg bw/day
No. of animals per sex per dose:
8 males and 16 females per group
Control animals:
yes, plain diet
Positive control:
no

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 (if feeding study):
- Food consumption: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
viability of sperms was checked
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 / F3 offspring:
number of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, but not described in detail

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy was performed on all animals with exception of the P0, P1 and P2 control and test groups

HISTOPATHOLOGY / ORGAN WEIGHTS
not specified
Postmortem examinations (offspring):
no specified
Statistics:
Numerical deviation from the control observations were evaluated by conventional statistical tests using P < 0.05 as the fiducial limit.
Reproductive indices:
Fertility index = number of pregnancies / number of matings x 100
Offspring viability indices:
Lactation index = number of weaned / number left to nurse x 100
Live birth index = number of pups born alive / number of pups born x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities were observed in the organs examined.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed the lack of viable sperm in the atrophied testes of all males at the 1170 ppm boron equivalent level of boric acid. Evidence was also found of decreased ovulation in the majority of the ovaries examined from the same level females sacrificed following the reproduction study.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
lack of viable sperms in the atrophied testes of all males at the high dose group
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The high dose group of male rats were sterile. An attempt to obtain litters by mating the treated females with the males fed only the basal diet was not successful.
The overall fertility indices for boric acid at levels of 177 and 350 ppm boron were significantly higher than those of the controls (see Tables 1). Higher lactation indices also occurred in rats fed at the same levels of boric acid. Live birth indices were within normal limits in the test groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
58.5 mg/kg bw/day (actual dose received)
System:
other: male and female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities were observed in the organs examined.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed the lack of viable sperm in the atrophied testes of all males at the 1170 ppm boron equivalent level of boric acid. Evidence was also found of decreased ovulation in the majority of the ovaries examined from the same level females sacrificed following the reproduction study.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
lack of viable sperms in the atrophied testes of all males at the high dose group
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The high dose group of male rats were sterile. An attempt to obtain litters by mating the treated females with the males fed only the basal diet was not successful.
The overall fertility indices for boric acid at levels of 177 and 350 ppm boron were significantly higher than those of the controls (see Table 1). Higher lactation indices also occurred in rats fed at the same levels of boric acid. Live birth indices were within normal limits in the test groups.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
58.5 mg/kg bw/day (actual dose received)
System:
other: male and female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Live birth indices were within normal limits in the test groups (mid and low; high dose group was sterile).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
weights of progeny was normal compared to those of controls
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities were observed in the organs examined from weanlings.
Histopathological findings:
not specified
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1a
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
other: no adverse effects described
Key result
Dose descriptor:
NOAEL
Generation:
F1b
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
other: no adverse effects described

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Live birth indices were within normal limits in the test groups (mid and low; high dose group was sterile).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
weights of progeny was normal compared to those of controls
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities were observed in the organs examined from weanlings.
Histopathological findings:
not specified
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F2a
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
other: no adverse effects described
Key result
Dose descriptor:
NOAEL
Generation:
F2b
Effect level:
17.5 mg/kg bw/day
Based on:
element
Remarks:
boron
Sex:
male/female
Basis for effect level:
other: no adverse effects described

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
58.5 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Table 1: Reproduction data for rats receiving boric acid at 117 and 350 ppm as boron equivalent

Index

Control

117 ppm

350 ppm

Control

117 ppm

350 ppm

 

P0 – F1a

P0 – F1b

Fertility index

62.5

87.5

81.3

60.0

87.5

75.0

Lactation index

56.3

96.2*

70.3*

58.8

85.6*

80.0*

Live birth index

98.4

96.0

97.2

99.1

99.4

100.0

 

P1 – F2a

P1 – F2b

Fertility index

81.3

93.8

93.8

80.0

93.8

93.8

Lactation index

48.3

79.2*

83.1*

92.1

81.0

98.0

Live birth index

97.8

100.0

99.4

98.6

99.4

97.9

 

P2 – F3a

P2 – F3b

Fertility index

68.8

100.0

87.5

68.8

93.8

93.8

Lactation index

91.5

82.5

86.5

89.7

86.7

87.9

Live birth index

100.0

99.5

97.9

100.0

99.0

98.8

* significant higher than control

Applicant's summary and conclusion

Conclusions:
In this three generation study with two litters per generation, were no adverse effects observed on the reproduction of rats receiving a diet containing boric acid at 117 (5.9 mg B/kg bw/day) and 350 (17.5 mg B/kg bw/day) ppm as boron equivalent. Litter size, weights of progeny and appearance were normal compared with those of the controls. The overall fertility indices for the two test compounds at levels of 177 and 350 ppm boron were significantly higher than those of the controls. Higher lactation indices also occurred in rats fed at the same levels of boric acid. Live birth indices were within normal limits in the test groups. No gross abnormalities were observed in the organs examined from either parents or weanlings. The high level test groups fed both borax and boric acid at 1170 ppm as boron equivalent were found to be sterile. An attempt to obtain litters by mating the treated females with the males fed only the basal diet was not successful. Microscopic examination revealed the lack of viable sperm in the atrophied testes of all males at the 1170 ppm (58.5 mg B/kg bw/day) boron equivalent level of boric acid. Evidence was also found of decreased ovulation in the majority of the ovaries examined from the same level females sacrificed following the reproduction study.