Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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)
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.
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
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: 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.
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.
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.
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
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.
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
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
116.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP complaint OECD guideline study, klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

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 (TNO Quality of Life, 2007). 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 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 are 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 16,5 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 group/ 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.


Short description of key information:
The NOAEL in rats is 116.5 and 40 mg/kg bw for fertility and parental toxicity, respectively.

Justification for selection of Effect on fertility via oral route:
Only study available.

Effects on developmental toxicity

Description of key information

Under these experimental conditions and results of the developmental toxicity rat study (OECD 414), the NOAEL for maternal parameters was considered to be 250 mg/kg/day. The NOAEL for the embryo-fetal development was considered to be 750 mg/kg/ day. Therefore, the substance is considered not to be teratogenic in the rat under these exprimental conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-01-13 to 2016-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study compliant with OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
2015-03-05
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: mean body weight of 292 g (range: 234 g to 360 g)
- Housing: The animals were individually housed in cages (Tecniplast 2154: polycarbonate with stainless steel lids, cage dimensions 48 x 26.5x21 cm; 940 cm2 ) containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen so as not to jeopardize gestation.
Each cage contained nylabone and rat hut for the environmental enrichment of the animals.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 3044117, (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 4 or 5 days before the beginning of the treatment period (arrival of females on Day 1 or 2 p.c.).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): : 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From:2016-01-28 To: 2016-02-25
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The correct amount of substance was weighed and put in the CMC 1% and strirred to obtained a homogenous suspension

VEHICLE
- Justification for use and choice of vehicle (if other than water): the substance is stable in CMC 1% .
- Concentration in vehicle: 0, 17, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): SLBK4363V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical technique: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Solvent and reagents:
+ Milli-Q water
+ Methanol for HPLC

Diluent 1: Milli-Q water
Diluent 2: Milli-Q water / Methanol (50v/50v)

Equipement:
+High Performance Liquid Chromatography (HPLC) system: Agilent Serie 1100
+Mass spectrometer API 2000
+ Balance Mettler Toledo
+ Automatic pipette biohit
+ Sofware: Analyst 1.4.2 (Agilent Technologie)

LOQ: 0.001 µg/mL

Chromatographic conditions:
+ Column: Kinetex C15 (Phenomenex), particle size = 2.6 µm, Lenght = 100 mm, inner diameter = 4.6 mm
+ Mobile phase: Milli-Q water/methanol (50/50) (v/v)
+ Elution mode: Isocratic
+ Flow rate: 0.4 mL/min
+ Software: analyst 1.4.2
+ Column temperature: 25°C
+ Injector temperature: Not controled
+Injection volume: 5µL
+ Needlewash: Methanol / Milli-Q water (50/50) (v/v)
+ Analysis time: 5 min
+ Retention time: 1.9 minutes

Results are expressed in mg/mL




Details on mating procedure:
The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c.
Duration of treatment / exposure:
The dose formulations were administered daily from Day 6 to Day 20 p.c. inclusive.
Frequency of treatment:
Once a day
Duration of test:
the animals were euthanazied on day 21 post coitum
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A previous OECD 421 study where Wistar rats were given the test item at 40, 116.5, 350 or 1000 mg/kg/day under a dose-volume of 2 mL/kg/day, during premating, mating, gestation and/or beginning of lactation periods.
At 1000 mg/kg/day, no females were treated as treatment was stopped in males after 3 days (10% of body weight loss).
At 350 mg/kg/day, two females died during the lactation period. There were also in females increase of water consumption, lower mean body weight gain in the second week of gestation, lower mean body weight during the last 2 weeks of gestation, lower food consumption in premating, gestation and mainly lactation. There were also 7/12 non-pregnant females, lower number of corpora lutea, of implantations and of live pups. During lactation, pup mortality and runts were noted at this dose.
At 116.5 mg/kg/day, the only test item-related finding in females was increase in the number of runts in pups.
At 40 mg/kg/day, there were no relevant test item-related effects.
A preliminary prenatal development study (CiToxLAB France/Study No. 43314 RSR) where Sprague Dawley rats were given the test item at 100, 500 or 1000 mg/kg/day from Days 6 to 20 p.c. in the same vehicle and at 5 mL/kg/day.
At 1000 mg/kg/day, 2/5 females were sacrificed on Day 9 or 12 p.c. for bad health condition, no food consumption and severe body weight loss. At necropsy both had many whitish areas on the heart. The three other females experienced piloerection and/or round back. Their food consumption was reduced in the first week of the treatment period and 2/3 females concomitantly lost body weight (-1 to -10%). There were no necropsy findings in surviving females, no external findings in fetuses and body weight of the two litters available was lower than in controls.
There were no obvious effects at 100 and 500 mg/kg/day, except maybe a dose-related but non statistically significant reduction of fetal body weight.

Therefore, 1000 mg/kg/day was considered to be too high and 500 mg/kg/day to low for the high dose level in the current main study. 750 mg/kg/day was thus selected as the high dose-level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 85 and 250 mg/kg/day).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature euthanasia.

FOOD CONSUMPTION
- The quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined:
The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy.

OTHER:
For apparently non-pregnant females, the absence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).

A gross evaluation of placentas was also undertaken. The placenta weight was recorded for each fetus and the ratio of fetal weight to placental weight was calculated.

Preservation of tissues
Macroscopic lesions observed in principal females (including those from the sacrificed prematurely female) were sampled and kept preserved in 10% buffered formalin. The corresponding tissues of five control animals were sampled and preserved.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

Fetal examinations:
Body weight of fetuses
The body weight of each fetus was recorded.

Sex of fetuses
The sex of each fetus was determined at the time of hysterectomy by visual assessment of anogenital distance and was confirmed by internal examination of sexual organs at detailed dissection of the soft tissues or at evisceration.


- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: No
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Indices:
Number of corpora lutea
Number of implantation sites
Pre-implantation loss (%)
Number of live fetuses
Number of dead fetuses
Number of early + late resorptions
Number of early resorptions
Number of late resorptions
Post-implantation loss (%)
Historical control data:
Yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 750 mg/kg/day:
- one premature sacrifice for bad health condition,
- isolated clinical signs (emaciated appearance and piloerection),
- minimal mean body weight loss in the first 3 days of treatment, followed by a tendency towards low mean body weight gains until Day 15 p.c.; low mean body weight from Day 9 p.c.,
- low mean food consumption throughout the entire treatment period,
- low mean carcass weight and mean net body weight change,

At 250 mg/kg/day:
- tendency towards low mean body weight gain up to Day 15 p.c.,
- slightly low mean food consumption in the first 3 days of treatment,
- low mean net body weight change from Day 6 p.c.

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Non-adverse effects:
At 750 mg/kg/day:
- low mean fetal body weight.
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Non-adverse effects:
At 750 mg/kg/day:
- Ossification delay,
- Increased incidences of fetal skeletal variations (ribs, head and metatarsals).

At 250 mg/kg/day:
- Increased incidences of fetal skeletal variations (ribs).

At 85 mg/kg/day:
- Increased incidences of fetal skeletal variations (ribs).
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Non-adverse effects:
At 750 mg/kg/day:
- Low mean fetal body weight and mean fetal body weight/placental weight ratio
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: None.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
Developmental effects observed:
no

Mean body weights and body weight changes (g) of pregnant females

 

Dose-level (mg/kg/day)

0

85

250

750

Body weight (g)

Day 6p.c.

293

290

292

293

Variation vs Control

 

-1

0

0

Day 9p.c.

308

303

301

291

Variation vs Control

 

-2

-2

-6

Day 12p.c.

329

322

321

306*

Variation vs Control

 

-2

-2

-7

Day 15p.c.

352

345

341

328**

Variation vs Control

 

-2

-3

-7

Day 18p.c.

397

391

383

373

Variation vs Control

 

-2

-4

-6

Day 21p.c.

446

439

428

415

Variation vs Control

 

-2

-4

-7

Body weight change (g)

Days 6 - 9p.c.

+15

+14

+9

-1#

Days 9 - 12p.c.

+21

+19

+20

+14

Days 12 - 15p.c.

+23

+23

+20

+18

Days 15 - 18p.c.

+45

+45

+42

+46

Days 18 - 21p.c.

+49

+48

+46

+42

Days 6 - 15p.c.

+59

+56

+49**

+36#

Days 6 - 21p.c.

+153

+149

+136

+124#

Statistically significant vs. controls: *: p<0.05, **: p<0.01, #: p<0.001

 

Mean food consumption (g/female/day) of pregnant

 

Dose-level (mg/kg/day)

0

85

250

750

. Days 6 - 9p.c.

25

25

22*

17#

Variation vs Control

 

0

-12

-32

. Days 9 - 12p.c.

27

26

25

19#

Variation vs Control

 

-4

-7

-30

. Days 12 - 15p.c.

29

29

27

25#

Variation vs Control

 

0

-7

-14

. Days 15 - 18p.c.

33

32

31

30

Variation vs Control

 

-3

-6

-9

. Days 18 - 21p.c.

34

33

31

29**

Variation vs Control

 

-3

-9

-15

Statistically significant vs. controls: *: p<0.05, **: p<0.01, #: p<0.001

 

Mean gravid uterus, carcass and net body weights (g)

 

Dose-level (mg/kg/day)

0

85

250

750

Gravid uterus weight (a)

103

103

98.4

96.3

Variation vs Control

 

0

-4

-7

Carcass weight (a)

344

336

330

319*

Variation vs Control

 

-2

-4

-7

Net body weight change from Day 6p.c.

50.6

45.9

37.9**

27.8#

Statistically significantvs.controls:*: p<0.05, **: p<0.01, #: p<0.001

 

Macroscopic post-mortem examination

 

Dose-level (mg/kg/day)

0

85

250

750

Right kidney: many yellowish raised foci

0

0

0

1

Right kidney: marked dilated renal pelvis

0

0

0

1

Right or left kidney: dilated pelvis

0

0

0

2

Right ureter: dilatation

0

0

0

2

Connective tissue, periovarian region: nodule(s), highly vascularised

0

0

0

1

Right mammary gland No. 6:
whitish palpable mass

0

0

0

1

Placentas fused, one implantation site only

0

1

0

0

Number of affected animals

0/22

1/24

0/24

6/23

 

Mean hysterectomy data per female

 

Dose-level (mg/kg/day)

0

85

250

750

HCD

study mean
[min-max]

Number of females with live fetuses on Day 21p.c.

22

24

24

23

Mean number of corpora lutea per animal

14.7

14.7

15.0

14.9

[14.4-16.6]

Mean number of implantation sites per animal

13.7

13.7

13.6

14.3

[12.9-14.6]

Pre-implantation loss (%)

6.7

8.4

9.1

4.3

[6.5-12.7]

Mean number of live fetuses per animal

12.9

12.8

12.6

12.7

[12.5-14.0]

Number of dead fetuses

0.0

0.0

0.0

0.0

[0.00-0.10]

Number of early + late resorptions

0.7

0.9

1.0

1.5

[0.17-0.80]

Number of early resorptions per animal

0.7

0.7

0.9

1.3

-

Number of late resorptions per animal

0.0

0.2

0.1

0.2

-

Post-implantation loss (%)

6.6

6.2

7.6

10.3

[1.5-6.2]

HCD: Historical control data (October 2014) of Sprague-Dawley rats from Janvier; -: not presented in HCD.

Mean sex ratio data per litter

 

Dose-level (mg/kg/day)

0

85

250

750

Male fetuses(%)

53.6

48.9

48.4

50.4

Mean placental and fetal body weights per litter (g)

 

Dose-level (mg/kg/day)

0

85

250

750

Fetal body weight (males + females)

5.78

5.86

5.60

5.44#

Variation vs Control

 

+1

-3

-6

Placental weight (males + females)

0.72

0.69

0.68

0.72

Variation vs Control

 

-4

-6

0

Fetal body weight/placental weight ratioa

8.33

8.75

8.51

7.85

Variation vs Control

 

+5

+2

-6

Statistically significantvs.controls:#: p<0.001; in italic: percentage differences vs.controls.

a: no statistics performed as not calculated by Reprotox software.

Fetal external variations

 

Dose-level (mg/kg/day)

0

85

250

750

HCD

study mean

maximum

Litters evaluated

22

24

24

23

Fetuses evaluated

283

307

303

293

Misshapen head, F (L)

 

0.3 (4.2)

0.3 (4.2)a

 

-

Protruding tongue, F (L)

 

0.3 (4.2)

0.7 (8.3)b

 

-

Paw hyperflexion, F (L)

 

 

0.3 (4.2)a

 

-

Malrotated limb, F (L)

 

 

 

0.3 (4.3)

-

Total of fetal external variations, F (L)

0 (0)

0.3 (4.2)

0.7 (8.3)

0.3 (4.3)

 

F: fetal incidence (%); L: litter incidence (%); a: fetus F22546-3; b: fetuses F22546-3 and F22547-02; HCD: Historical control data in % (October 2014) of Sprague-Dawley rats; -: none in HCD

Fetal external malformations

 

Dose-level (mg/kg/day)

0

85

250

750

HCD

study mean

maximum

Litters evaluated

22

24

24

23

Fetuses evaluated

283

307

303

293

Anasarca, F (L)

 

 

0.3 (4.2)a

 

-

Short mandible, F (L)

0.4 (4.5)

 

0.3 (4.2)a

 

-

Short snout, F (L)

 

 

0.7 (8.3)b

 

-

Maxillary micrognathia, F (L)

 

 

0.7 (8.3)b

 

-

Ectrodactyly, F (L)

 

 

0.3 (4.2)a

 

-

Short digit(s), F (L)

 

 

0.3 (4.2)a

 

-

Micromelia, F (L)

 

 

0.3 (4.2)a

 

-

Hemimelia, F (L)

 

 

0.3 (4.2)a

 

-

Gastroshisis, F (L)

 

 

0.3 (4.2)a

 

0.4 (5.6)

Bent tail, F (L)

 

 

0.3 (4.2)a

 

-

Anal aresia, F (L)

 

 

0.3 (4.2)a

 

-

Short trunk, F (L)

 

 

0.3 (4.2)a

 

-

Total of fetal external malformations, F (L)

0.4 (4.5)

0 (0)

0.7 (8.3)a

0 (0)

 

F: fetal incidence (%); L: litter incidence (%); a: fetus F22546-3; b: fetuses F22546-3 and F22547-02; HCD: Historical control data in % (October 2014) of Sprague-Dawley rats; -: none in HCD.

Conclusions:
Based on the observations during the study, the substance can be considered as non teratogenic.
Executive summary:

In a GLP compliant developmental toxicity test, performed according to OECD Guideline 414, time-mated female Sprague-Dawley rats

were orally exposed to potassium tetrafluoroborate. 1% carboxymethylcellulose solution in water (CMC) was used as vehicle.

The test item, Potassium Tetrafluoroborate (batch No. BWF4103) was administered by oral gavage daily from Day 6 to Day 20p.c. inclusive at doses of 85, 250 or 750 mg/kg/day.

The toxicologically significant test item treatment effects were the following:

At 750 mg/kg/day

- one premature sacrifice for bad health condition,

- isolated clinical signs (emaciated appearance and piloerection),

-minimal mean body weight loss in the first 3 days of treatment, followed by a tendency towards  low mean body weight gains until Day 15p.c.

- low mean body weight from Day 9p.c

-low mean food consumption throughout the entire treatment period,

-low mean carcass weight and mean net body weight change,

- low mean fetal body weight and mean fetal body weight/placental weight ratio, together with ossification delay

- increased incidences of fetal skeletal variations (ribs, head and metatarsals).

 

At 250 mg/kg/day:

      - tendency towards low mean body weight gain up to Day 15p.c.,

      - slightly low mean food consumption in the first 3 days of treatment,

      - low mean net body weight change from Day 6p.c.,

      - increased incidences of fetal skeletal variations(ribs).

 

At 85 mg/kg/day:

      - increased incidences of fetal skeletal variations(ribs). 

Under the experimental conditions and results of this study, the NOAEL for maternal parameters was considered to be 250 mg/kg/day. The NOAEL for the embryo-fetal development was considered to be 750 mg/kg/ day.

Therefore, the substance is considered not to be teratogenic in this species under these exprimental conditions.

Endpoint:
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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
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 female swere 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) roden diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, 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
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
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.
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
Duration of treatment / exposure:
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
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.
Maternal 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.

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), organs and tissues showing macroscopic abnormalities.
Fetal examinations:
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
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. Histopathological changes were evaluated by Fisher's exact probability test.
Indices:
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

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
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

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
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
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
Mean body weight and/or body weight change of the female animals of 350 mg/kg bw/day group were statistically significantly decreased during several periods of the study. 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
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 PERFORMANCE
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/kg bw/day groups, respectively. The mating index was 100% in all KBF4-treated groups and 92% in the control group. The female fecundity index and female 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 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.

GROSS PATHOLOGY
At scheduled necropsy no treatment-related gross changes were observed.

HISTOPATHOLOGY
Microscopic examination did not reveal treatment related histopathological changes in any of the sampled organs and tissues.

OTHER FINDINGS
Effects on water consumption were observed (not exactly measured) in the 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.
Dose descriptor:
NOAEL
Effect level:
116.5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
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.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
The NOAEL in rats is 116.5 and 40 mg/kg bw for maternal toxicity and developmental toxicity, respectively.
Executive summary:

In a GLP compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, Wistar rats were orally exposed to potassium tetrafluoroborate. 1% carboxymethylcellulose solution in water (CMC) was used as vehicle. Groups of 12 rats were dosed daily with 0, 40, 116,5 and 350 mg KBF4/kg body weight during 4 weeks premating, mating, gestation and up to day 4 of lactation. 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 350 mg KBF4/kg body weight group from week 3 onwards. Mean body weight and or body weight change of the animals of the 350 mg KBF4/kg body weight were statistically significantly decreased during several periods of the study. Food consumption of the 350 mg KBF4/kg body weight group was statistically significantly decreased during several periods of the study. 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 and female 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 fecundity index and female 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 are did not reveal any treatment related abnormalities. 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. Based on the effects on mortality, body weight and food consumption in the 350 mg KBF4 group/ kg body weight group, the NOAEL for maternal toxicity is 116.5 mg KBF4/kg body weight/day. The NOAEL for developmental toxicity is 40 mg KBF4/kg body weight based on the increased pup mortality (PN 1-4) in the 350 mg KBF4/kg body weight group and the increased number of runts in the 116.5 and 350 mg KBF4/kg body weight groups.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP complaint OECD guideline study, klimisch 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP compliant reproduction/developmental toxicity screening test, performed according to OECD Guideline 421, Wistar rats were orally exposed to potassium tetrafluoroborate (TNO Quality of Life, 2007). 1% carboxymethylcellulose solution in water (CMC) was used as vehicle. Groups of 12 rats were dosed daily with 0, 40, 116.5 and 350 mg KBF4/kg body weight during 4 weeks premating, mating, gestation and up to day 4 of lactation (females). 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 350 mg KBF4/kg body weight group from week 3 onwards. Mean body weight and or body weight change of the animals of the 350 mg KBF4/kg body weight were statistically significantly decreased during several periods of the study. Food consumption of the 350 mg KBF4/kg body weight group was statistically significantly decreased during several periods of the study. 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 and female 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 fecundity index and female 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 are did not reveal any treatment related abnormalities. 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. Based on the effects on mortality, body weight and food consumption in the 350 mg KBF4 group/ kg body weight group, the NOAEL for maternal toxicity is 116.5 mg KBF4/kg body weight/day. The NOAEL for developmental toxicity is 40 mg KBF4/kg body weight based on the increased pup mortality (PN 1-4) in the 350 mg KBF4/kg body weight group and the increased number of runts in the 116.5 and 350 mg KBF4/kg body weight groups.

In a GLP compliant developmental toxicity test, performed according to OECD Guideline 414,time-mated female Sprague-Dawley rats

were orally exposed to potassium tetrafluoroborate. 1% carboxymethylcellulose solution in water (CMC) was used as vehicle.

The test item, Potassium Tetrafluoroborate (batch No. BWF4103) was administered by oral gavage daily from Day 6 to Day 20p.c. inclusive at doses of 85, 250 or 750 mg/kg/day.

The toxicologically significant test item treatment effects were the following:

At 750 mg/kg/day

- one premature sacrifice for bad health condition,

- isolated clinical signs (emaciated appearance and piloerection),

-minimal mean body weight loss in the first 3 days of treatment, followed by a tendency towards  low mean body weight gains until Day 15p.c.

- low mean body weight from Day 9p.c

-low mean food consumption throughout the entire treatment period,

-low mean carcass weight and mean net body weight change,

- low mean fetal body weight and mean fetal body weight/placental weight ratio, together with ossification delay

- increased incidences of fetal skeletal variations (ribs, head and metatarsals).

 

At 250 mg/kg/day:

      - tendency towards low mean body weight gain up to Day 15p.c.,

      - slightly low mean food consumption in the first 3 days of treatment,

      - low mean net body weight change from Day 6p.c.,

      - increased incidences of fetal skeletal variations(ribs).

 

At 85 mg/kg/day:

      - increased incidences of fetal skeletal variations(ribs). 

Under the experimental conditions and results of this study, the NOAEL for maternal parameters was considered to be 250 mg/kg/day. The NOAEL for the embryo-fetal development was considered to be 750 mg/kg/ day.

Therefore, the substance is considered not to be teratogenic in this species under these exprimental conditions.

Justification for classification or non-classification

Based on the absence of adverse effects on fertility and of developmental toxicity in the absence of parental toxicity, classification of the test material for effects on fertility or developmental toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.