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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
2015-03-05
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name: Potassium Tetrafluoroborate
CAS: 14075-53-7

Test animals

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

Administration / exposure

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).

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.