Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): potassium tetrafluoroborate
- Physical state: white powder
- Analytical purity: 98.9%
- Lot/batch No.: BWF41213
- Expiration date of the lot/batch: 13 December 2009
- Storage condition of test material: ambient temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany, SPF-bred colony
- Age at study initiation: (P) 8-9 wks
- Weight at study initiation: (P) within 20% of the mean weight for each sex
- Housing: in macrolon cages with wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment. During the premating period, the animals were housed in groups of 4/sex. For mating, 1 male and 1 female were housed together. Mated femaleswere housed individually in macrolon cages, which were then placed in another cage rack. After delivery, the cage containing the dam with litter were transferred to another cage rack
- Diet: cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum
- Water: tap water in polypropylene bottles, cleaned weekly and filled as needed, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% solution in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was suspended in 1% CMC. The dosing solutions were prepared weekly and stored at 2-10 ºC. The miscibility of the test substance in vehicle were checked by visual inspection before the start of the study.

VEHICLE
- Concentration in vehicle: 0, 20, 58.2, 175 and 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 101K0185
- Purity: 1% solution in water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until pregnancy occurred
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually for the birth and rearing of the pups until day 4 of lactation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the stability, homogeneity and content of the test substance in the vehicle was conducted, by quantitative determination of the level of potassium tetrafluoroborate by determination of boron using ICP-AES and determination of fluoride using GC-FID with headspace.
Duration of treatment / exposure:
Males: during 4 weeks premating period and during the mating period for at least 5 weeks until sacrifice. Females: during a 2-week premating period, during the mating, gestation and lactation period until sacrifice. Animals were not dosed on the day of necropsy.
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: ca. 13-14 weeks (males); ca. 11-12 weeks (females)
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 116.5 and 350 mg/kg bw/day (adjusted from 116.5, 350 and 1000 mg/kg bw/day due to body weight loss in high-dose animals)
Basis:
actual ingested
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: computer randomization proportionally to body weight.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On Saturdays and Sundays only one check per day was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before the time of dosing (randomization) and on the first day of dosing and weekly thereafter during the premating period. In addition, body weights of the male animals were measured on day 3 and 5 of the study. Males were weighed approximately weekly during the mating period until sacrifice. Females were weighed approximately weekly during mating and mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation. All animals were weighed on the day of sacrifice.

FOOD CONSUMPTION: Yes
Food consumption of male rats was measured approx. weekly, except during the mating period. In addition, food consumption of the male animals was measured on day 3 and 5 of the study, except for male animals of lowest dose group.
Food consumption of female rats was measured weekly during the premating period and during the gestation period from gestation days 0-7, 7-14 and 14-21, and once during the lacation period from day 1 to 4.

WATER CONSUMPTION: Yes
During daily observations, until day 5 of gestation.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymides weight, sperm count in testes, sperm count in epididymides, epididymal sperm motility and sperm morphology.
In addition, reproductive organs of males that fail to sire (did not mate or mated female was not pregnant) of the 40 and 116.5 mg/kg bw/day were macroscopically examined.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, number of litters lost entirely, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all male animals of the 1000 mg/kg bw/day group were sacrified on day 7 of the study after 3 daily dosages and 5 days of recovery. Males from other groups were euthanised after approximately 5 weeks of exposure.
- Maternal animals: sperm-positive females that turned out to be non-pregnant were killed 24-26 days after copulation. Females that became pregnant were sacrificed at day 4 of lactation.

GROSS NECROPSY
Animals were examined grossely for macroscopic changes.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of the following organs were preserved: ovaries (after counting of the corpora lutea), uterus (after counting of the implantation sites), testes, epididymides, seminal vehicles, prostate, organs and tissues showing macroscopic abnormalities.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrified at 4 days of age.
Macroscopic examination was performed on stillborn pups and pups that died between postnatal days 1-4.
Statistics:
Clinical findings were evaluated by Fisher's probability test. Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. Fisher's exact probability test were used to evaluate the number of mated and pregnant females and females with live pups. Number of implantation sites, live and dead pups were evaluated by the Mann Whitney U-test. Sperm parameters were evaluated by ANOVA followed by Dunnet's multiple comparison tests (epididymal sperm count and numerical sperm motility parameters) or by Kruskal-Wallis nonparametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology). Histopathological changes were evaluated by Fisher's exact probability test.
Reproductive indices:
The following data were collected:
- number of succesful copulations (number of females mated)
- number of males that became sire
- number of pregnant females as demonstrated by the presence of implantation istes observed at autopsy.
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups

The following indices were calculated:
Pre-coital time = time between the start of mating and successful copulation
Duration of gestation = time between gestation day 0 and day of delivery
Mating index = (number of females mated/number of females placed with males) x 100
Male fertility index = (number of males that became sire/number of males placed with females) x 100
Female fertility index = (number of pregnant females/number of females placed with males) x 100
Female fecundity index = (number of pregnant females/number of females mated) x 100
Gestation index = (number of females with live pups/number of females pregnant) x 100
Offspring viability indices:
The following data were collected:
- number of pups delivered (live and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of corpora lutea
- number of male pups at day n
- number of implantation sites
- number of lost implantations
- litter size

The following indices were calculated:
Live birth index = (number of pups born alive/number of pups born) x 100
Pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
Sex ratio day n = (number of live male pups on day n/number of live pups on day n) x 100
Number of lost implantations = number of implantation sites-number of pups born alive
Pre-implantation loss = [(number of corpora lutea-number of implantation sites/number of corpora lutea] x 100
Post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Two female animals of the 350 mg/kg bw/day died during lactation period
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
corpora lutea and implantation sites in the 350 mg KBF4/kg body weight group.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
decreased number of pregnant females, corpora lutea and implantation sites in the 350 mg KBF4/kg body weight group.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Two female animals of the 350 mg/kg bw/day died during lactation period. Daily clinical observations during the study did not reveal any remarkable findings in animal appearance, general condition or behaviour among the dosing and control groups.

BODY WEIGHT (PARENTAL ANIMALS)
Twelve male animals of the 1000 mg/kg bw/day group lost ca. 10% (30 g) of their initial body weight on day 0 after 3 daily dosages. It was decided, after the consultation with the sponsor, to stop dosing these male animals and to keep these animals in the study without treatment up to day 7, to study the reversibility of this effect. Mean body weight of the animals of day 7 was comparable to their body weight on day 0.
Mean body weight and/or body weight change of the male and female animals of 350 mg/kg bw/day group were statistically significantly decreased during several periods of the study. Mean body weight of the male animals of the 350 mg/kg bw/day group was statistically significantly decreased from day 3 until sacrifice. Mean body weight change of the male animals of this group was statistically significantly decreased between days 0-3, 3-5, 7-14 and 21-28. Mean body weight of the male animals of the 116.5 mg/kg bw/day group was statistically significantly decreased from day 28 until sacrifice. Mean body weight change of the male animals of this group was statistically significantly decreased between days 21-28. No decreased in body weight and body weight change was observed in the male animals of the 40 mg/kg bw/day group. During the premating period, no effect on body weight or body weight change was observed in female animals treated with KBF4. Mean body weight of the female animals of the 350 mg/kg bw/day group was statistically significantly decreased from gestation day 7-21. Mean body weight change of the female animals of this group was statistically significantly decreased from gestation days 7-14. Mean body weight of the female animals of this group was statisitcally significantly decreased on lactation day 1. During the lactation period no effect was observed on mean body weight change of the KBF4-treated females.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption of the male animals of the 1000 mg KBF4/kg body weight group was statistically significantly decreased (5.45 g/animal/day versus 19.65 g/animal in the control group) from day 0-3. Animals of this group were not longer dosed from day 3 to 7; food consumption of the animals was back to normal or even higher from day 5 to 7. Food consumption of the male animals of the 350 mg KBF4/kg body weight group was statistically significantly decreased during the premating period. Food consumption of the animals of the 116.5 mg KBF4/kg body weight group was comparable to the control group except for the statistical significant difference between dat 7 and 14. Food consumption of the female animals of the 350 mg KBF4/kg body weight group was statistically significantly decreased from day 14-21 of the premating period, during GD 0-14 (g/animal/day) and GD 0-7 (g/kg/day) and day 1-4 of lactation (g/animal/day). One animal of this group did not eat any food from lactation day 1-4. Furthermore, no remarkable differences were observed in the KBF4-treated groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No differences were observed in the motility, count and morphology of the epididymal sperm at scheduled necropsy.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In each group 12 females were placed with males. All females of the KBF4-treated groups and 11 females of the control group were mated within 4 days. One female of the control group gained approx. 20 g weight during the first week of the mating period. For that reason it was supposed the positive smear was missed and the female was assumed to be mated and removed from the male; this female appeared to be not pregnant. The number of pregnant females, the number of females with live born pups and the number of males that became sires amounted to 8, 9, 9, and 5 for the control, 40, 116.5 and 350 mg/kg bw/day, respectively. The mating index was 100% in all KBF4-treated groups and 92% in the control group. The female fecundity index, female fertility index and male fertility index were comparable between the control, 40 and 116.5 mg/kg bw/day groups and ranged from 67-75%. In the 350 mg/kg bw/day group, the fecundity index, female fertility index and male fertility index was 42%. The gestation index was 100% in all groups. The duration of gestation was comparable between the groups. The number of corpora lutea and implantation sites was statistically significantly decreased in the 350 mg/kg bw/day group; no effect was observed in other treated groups when compared to the control group. Pre- and post-implantation loss was comparable in all groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute and relative testes and epididymides weights were comparable in all groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At scheduled necropsy no treatment-related gross changes were observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopic examination did not reveal treatment related histopathological changes in any of the sampled organs and tissues (epididymides, testes, uterus and ovaria).

OTHER FINDINGS (PARENTAL ANIMALS):
Effects on water consumption were observed (not exactly measured) in the male and female animals of the 350 mg/kg bw/day group from week 3 onwards. In consultation with the study director it was decided to stop registration of water consumption after day 5 of gestation.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
116.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the decreased number of pregnant females, corpora lutea and implantations sites at the next dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of litters with live born pups was 8, 9, 9 and 5 for the control, 40, 116.5 and 350 mg/kg bw/day groups, respectively. The number of live pups per litter on postnatal day 1 in the control, 40, 116.5 and 350 mg/kg bw/day groups was 11.9, 12.1, 11.8 and 9.4, respectively. Pup mortality on postnatal day 4 amounted to 3, 13, 3 and 29 (incidences 3.2, 12, 2.8 and 62%) in the control, 40, 116.5 and 350 mg/kg bw/day groups, respectively; in the 350 mg/kg bw/day group the mortality was statistically significantly increased. The death of the pups of two dams (10 and 11 pups, respectively) could be secondary to the death of the dams on postnatal days 2 and 3, respectively. In the control, 40, 116.5 and 350 mg/kg bw/day groups 0(8), 1(8), 0(9) and 3(2) litters, respectively, were lost entirely between postnatal days 1-4 (between brackets the number of litters with live pups on postnatal day 4). The number of live pups per litter on postnatal day 1 was 11.9, 12.1, 11.8 and 9.4 in the control, 40, 116.5 and 350 mg/kg bw/day groups, respectively, and on postnatal day 4 the number of pups was 11.5, 12.0, 11.4 and 9.0 in the control , 40, 116.5 and 350 mg/kg bw/day groups, respectively. The number of live pups per litter of the 350 mg/kg bw/day weight group was statistically significantly decreased on postnatal day 1. On postnatal day 4 only pups of 2 litters in the high-dose group were alive. The viability index on postnatal days 1-4 was 97, 88, 97 and 38% for the 0, 40, 116.5 and 350 mg/kg bw/day groups, respectively.

BODY WEIGHT (OFFSPRING)
No differences were observed for pup weight and pup weight changes on postnatal days 1 and 4. On postnatal day 1 the number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was statistically significantly increased in the 116.5 and 350 mg/kg bw/day groups. In addition, the number of runts was statistically significantly increased in the 116.5 mg/kg bw/day group on postnatal day 4.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic observations in stillborn pups and pups that died between postnatal days 1-4 did not reveal any treatment related abnormalities.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
mortality
other: increased number of runts in the 116.5 and 350 mg/kg bw/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL in rats is 40, 116.5 and 40 mg/kg bw for parental, fertility and developmental toxicity, respectively.
Executive summary:

In a GLP compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, Wistar rats of both sexes were orally exposed to potassium tetrafluoroborate. 1% carboxymethylcellulose solution in water (CMC) was used as vehicle. Groups of 12 rats per sex were dosed daily with 0, 40, 116,5 and 350 mg KBF4/kg body weight for up to approx. 35 days (males) or during 4 weeks premating, mating, gestation and up to day 4 of lactation (females). Initially a group of male rats was dosed with 1000 mg KBF4/kg body weight. These animals showed a weight loss of approx. 10% after 3 daily dosages. For that reason this group was replaced by the 40 mg KBF4/kg body weight. Two female animals of the 350 mg KBF4/kg body weight died during the lactation period. Daily clinical observations during the study did not reveal any remarkable findings in animals' appearance, general condition or behavior among the dosing and control groups. Effects on water consumption were observed (not exactly measured) in the male and female animals of the 350 mg KBF4/kg body weight group from week 3 onwards. Mean body weight and or body weight change of the male and female animals of the 350 mg KBF4/kg body weight were statistically significantly decreased during several periods of the study. Mean bodyweight or bodyweight change of the 116,5 mg KBF4/kg body weight group was only statistically significantly decreased in the male animals from day 21 onwards. Food consumption of the 350 mg KBF4/kg body weight group was statistically significantly decreased in male and female animals during several periods of the study. Food consumption of the male animals of the 116.5 mg KBF4/kg body weight group was statistically significantly decreased from day 7 to 14. In each group 12 females were placed with males. All females of the KBF4-treated groups and 11 females of the control group were mated within 4 days. The number of pregnant females, the number of females with live born pups and the number of males that became sires amounted to 8, 9, 9 and 5 for the control, 40, 116.5 and 350 mg KBF4/kg body weight groups, respectively. The mating index was 100% in all KBF4-treated groups and 92% in the control group. The female fecundity index, female fertility index and male fertility index were comparable between the control, 40 and 116.5 mg KBF4/kg body weight groups and ranged from 67-75%. In the 350 mg KBF4/kg body weight group, the fecundity index, female fertility index and male fertility index was 42%.The gestation index was 100% in all groups. The duration of gestation was comparable between the groups. The number of corpora lutea and implantation sites was statistically significantly decreased in the 350 mg KBF4/kg body weight group; no effect was observed in the other KBF4-treated groups when compared to the control group. Pre- and post-implantation loss was comparable in all groups. The number of litters with live born pups was 8, 9, 9 and 5 for the control, 40, 116.5 and 350 mg KBF4/kg body weight groups, respectively. The number of live pups per litter on post natal day (PN) 1 in the control, 40, 116.5 and 350 mg KBF4/kg body weight groups was 11.9, 12.1, 11.8 and 9.4, respectively. Pup mortality on PN 4 was comparable in all groups except for the mid-dose group and amounted to 3, 13, 3, 29 (incidences 3.2, 12, 2.8 and 62%) in the control, 40, 116.5 and 350 mg KBF4/kg body weight groups, respectively; in the 350 mg KBF4/kg body weight group the mortality was statistically significantly increased. The death of the pups of dam C63 (10 pups and C67 (11 pups) may be secondary to the death of the dams on PN 2 and 3, respectively. In the control, 40, 116,5 and 350 mg KBF4/kg body weight groups 0(8), 1(8), 0(9) and 3(2) litters, respectively were lost entirely between PN 1-4 (between brackets the number of litters with live pups on PN 4). The number of live pups per litter on PN 1 was 11.9, 12.1, 11.8 and 9.4 in the control, 40, 116.5 and 350 mg KBF4/kg body weight groups, respectively and on PN 4 the number of pups was 11.5, 12.0, 11.4 and 9.0 in the control, 40, 116.5 and 350 mg KBF4/kg body weight groups, respectively. The number of live pups per litter of the 350 mg KBF4/kg body weight group was statistically significantly decreased on PN 1. On PN 4 only pups of 2 litters of the mid-dose group were alive. No difference was observed in the sex ratio between the groups. No differences were observed on pup weight and pup weight changes on PN 1 and 4. On PN 1 the number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was statistically significantly increased in the 116.5 and 350 mg KBF4/kg body weight groups. In addition, the number of runts was statistically significantly increased in the 116.5 KBF4/kg body weight group on PN 4. Macroscopic observations in stillborn pups and pups that died between PN 1 -4 did not reveal any treatment related abnormalities. No differences were observed in the motility, count and morphology of the epididymal sperm at scheduled necropsy. Absolute and relative testes and epididymides weights were comparable in all groups. Terminal body weight of the male animals of the 40 and 116.5 mg KBF4/kg body weight groups was statistically significantly decreased. At scheduled necropsy no treatment related gross changes were observed. Microscopic examination did not reveal treatment related histopathological changes in any of the sampled organs and tissues (epididymides, testes, uterus and ovaria). Based on the effects on mortality in the 350 mg KBF4/kg body weight group, (terminal) body weight and food consumption in the 116.5 and 350 mg KBF4/kg body weight groups, the NOAEL for parental toxicity is 40 mg KBF4/kg body weight/day. The NOAEL for fertility in the parental animals is 116.5 mg KBF4/kg body weight, based on the decreased number of pregnant females, corpora lutea and implantation sites in the 350 mg KBF4/kg body weight group.